Production of isoprenoids

ABSTRACT

Methods for producing an isoprenoid are provided. A plurality of bacterial or fungal host cells is obtained. These cells comprise a heterologous nucleic acid encoding one or more enzymes of a mevalonate pathway for making isopentenyl pyrophosphate. Expression of the one or more enzymes is under control of at least one heterologous transcriptional regulator. The mevalonate pathway comprises (i) an enzyme that condenses acetoacetyl-CoA with acetyl-CoA to form HMG-CoA, (ii) an enzyme that converts HMG-CoA to mevalonate, (iii) an enzyme that phosphorylates mevalonate to mevalonate 5-phosphate, (iv) an enzyme that converts mevalonate 5-phosphate to mevalonate 5-pyrophosphate, and (v) an enzyme that converts mevalonate 5-pyrophosphate to isopentenyl pyrophosphate. The host cells are cultured in a medium under conditions that are suboptimal as compared to conditions for the maximum growth rate. Temperature is maintained at a level below that which would provide for a maximum specific growth rate for the host cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/638,771, filed Dec. 15, 2009, which is a continuation of U.S. patentapplication Ser. No. 11/754,235, filed May. 25, 2007, now issued as U.S.Patent No. 7,659,097, which claims priority to U.S. ProvisionalApplication Nos. 60/808,989, filed May 26, 2006 and 60/870,592 filed onDec. 18, 2006, which are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION

Isoprenoids are ubiquitous in nature. They comprise a diverse family ofover 40,000 individual products, many of which are vital to livingorganisms. Isoprenoids serve to maintain cellular fluidity, electrontransport, and other metabolic functions. A vast number of natural andsynthetic isoprenoids are useful as pharmaceuticals, cosmetics,perfumes, pigments and colorants, fungicides, antiseptics,nutraceuticals, and fine chemical intermediates.

An isoprenoid product is typically composed of repeating five carbonisopentenyl diphosphate (IPP) units, although irregular isoprenoids andpolyterpenes have been reported. In nature, isoprenoids are synthesizedby consecutive condensations of their precursor IPP and its isomerdimethylallyl pyrophosphate (DMAPP). Two pathways for these precursorsare known. Eukaryotes, with the exception of plants, generally use themevalonate-dependent (MEV) pathway to convert acetyl coenzyme A(acetyl-CoA) to IPP, which is subsequently isomerized to DMAPP.Prokaryotes, with some exceptions, typically employ only themevalonate-independent or deoxyxylulose-5-phosphate (DXP) pathway toproduce IPP and DMAPP. Plants use both the MEV pathway and the DXPpathway. See Rohmer et al. (1993) Biochem. J. 295:517-524; Lange et al.(2000) Proc. Natl. Acad. Sci. USA 97(24):13172-13177; Rohdich et al.(2002) Proc. Natl. Acad. Sci. USA 99:1158-1163.

Traditionally, isoprenoids have been manufactured by extraction fromnatural sources such as plants, microbes, and animals. However, theyield by way of extraction is usually very low due to a number ofprofound limitations. First, most isoprenoids accumulate in nature inonly small amounts. Second, the source organisms in general are notamenable to the large-scale cultivation that is necessary to producecommercially viable quantities of a desired isoprenoid. Third, therequirement of certain toxic solvents for isoprenoid extractionnecessitates special handling and disposal procedures, and thuscomplicating the commercial production of isoprenoids.

The elucidation of the MEV and DXP metabolic pathways has madebiosynthetic production of isoprenoids feasible. For instance, microbeshave been engineered to overexpress a part of or the entire mevalonatepathway for production of an isoprenoid named amorpha-4,11-diene (U.S.Pat. Nos. 7,172,886 and 7,192,751) Other efforts have focused onbalancing the pool of glyceraldehyde-3-phosphate and pyruvate, or onincreasing the expression of 1-deoxy-D-xylulose-5-phosphate synthase(dxs) and IPP isomerase (idi). See Farmer et al. (2001) Biotechnol.Prog. 17:57-61; Kajiwara et al. (1997) Biochem. J. 324:421-426; and Kimet al. (2001) Biotechnol. Bioeng. 72:408-415.

Nevertheless, given the very large quantities of isoprenoid productsneeded for many commercial applications, there remains a need forexpression systems and fermentation procedures that produce even moreisoprenoids than available with current technologies. Optimalredirection of microbial metabolism toward isoprenoid productionrequires that the introduced biosynthetic pathway is properly engineeredboth to funnel carbon to isoprenoid production efficiently and toprevent build up of toxic levels of metabolic intermediates over asustained period of time. The present invention addresses this need andprovides related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for a robustproduction of isoprenoids by the use of isopentenyl pyrophosphatepathway enzymes that are under the control of at least one heterologousregulator or fermentation conditions, either alone or in combination.Non-limiting examples of suitable isoprenoids include: hemiterpenes(derived from 1 isoprene unit) such as isoprene; monoterpenes (derivedfrom 2 isoprene units) such as myrcene; sesquiterpenes (derived from 3isoprene units) such as amorpha-4,11-diene; diterpenes (derived fromfour isoprene units) such as taxadiene; triterpenes (derived from 6isoprene units) such as squalene; tetraterpenes (derived from 8isoprenoids) such as β-carotene; and polyterpenes (derived from morethan 8 isoprene units) such as polyisoprene.

In one aspect, a method of producing an isoprenoid involves the steps of(a) obtaining a plurality of host cells that comprise an enzymaticpathway for making isopentenyl pyrophosphate wherein the all of thepathway enzymes are under control of at least one heterologoustranscriptional regulator; and (b) culturing the host cells in a mediumunder conditions that are suboptimal as compared to conditions thatwould provide for a maximum specific growth rate for the host cells. Insome embodiments, the pathway is the mevalonate pathway. In otherembodiments, the pathway is the DXP pathway. In other embodiments, theat least one heterologous transcriptional regulatory sequence isinducible. In other embodiments, the pathway enzymes are under controlof a single transcriptional regulator. In other embodiments, the pathwayenzymes are under control of multiple heterologous transcriptionalregulators.

In some embodiments, the pathway comprises a nucleic acid sequenceencoding a mevalonate pathway enzyme from a prokaryote having anendogenous mevalonate pathway. Exemplary prokaryotes having anendogenous mevalonate pathway include but are not limited to the genusEnterococcus, the genus Pseudomonas, and the genus Staphylococcus. Inone embodiment, the mevalonate pathway enzyme is selected fromacetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, and mevalonatekinase. In another embodiment, the heterologous nucleic acid sequenceencodes a Class II HMG-CoA reductase.

In another embodiment, the host cells are cultured in a medium whereinthe nutrient and/or temperature level is maintained at a level belowthat which would provide for the maximum specific growth rate for thehost cells. In another embodiment, the host cells are cultured in amedium where the carbon source is maintained at a level to provide forless than about 90%, 75%, 50%, 25%, 10%, or anywhere between 90% and 10%of the maximum specific growth rate. In another embodiment, the hostcells are cultured in a medium where the nitrogen source is maintainedat a level to provide for less than about 90%, 75%, 50%, 25%, 10%, oranywhere between 90% and 10% of the maximum specific growth rate. Inanother embodiment, the host cells are cultured in a medium where thetemperature is maintained at a level to provide for less than about 90%,75%, 50%, 25%, 10% or anywhere between 90% and 10% of the maximumspecific growth rate. In another embodiment, the medium temperature ismaintained at least about 2° C., 4° C., 5° C., 6° C., 8° C., 10° C., 15°C., or 20° C. below the temperature that would provide for the maximumspecific growth rate.

In yet another embodiment, a method of producing an isoprenoid orisoprenoid precursor comprises the steps of (i) performing afermentation reaction comprising a fermentation medium and a pluralityof genetically modified host cells that produce the isoprenoid underconditions such that (a) the fermentation medium is kept at atemperature lower than that which would provide for a maximum specificgrowth rate of said host cells; (b) the fermentation medium comprises acarbon source present in an amount that is lower than that which wouldprovide for a maximum specific growth rate of the host cells; and/or (c)the fermentation medium comprises a nitrogen source present in an amountthat is lower than that which would provide for a maximum specificgrowth rate of the host cells; (ii) recovering the isoprenoid producedunder one or more conditions set forth in (a) through (c). In oneaspect, the isoprenoid is produced under at least two of the conditionsset forth in (a) through (c). In another aspect, the isoprenoid isproduced under all of the conditions set forth in (a) through (c).

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of the mevalonate (“MEV”) pathwayfor the production of isopentenyl pyrophosphate (“IPP”).

FIG. 1B is a schematic representation of the 1-deoxy-D-xylulose5-diphosphate (“DXP”) pathway for the production of isopentenylpyrophosphate (“IPP”) and dimethylallyl pyrophosphate (“DMAPP”). Dxs is1-deoxy-D-xylulose-5-phosphate synthase; Dxr is1-deoxy-D-xylulose-5-phosphate reductoisomerase (also known as IspC);IspD is 4-diphosphocytidyl-2C-methyl-D-erythritol synthase; IspE is4-diphosphocytidyl-2C-methyl-D-erythritol synthase; IspF is2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; IspG is1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG); and ispHis isopentenyl/dimethylallyl diphosphate synthase.

FIG. 2 is a schematic representation of the conversion of isopentenylpyrophosphate (“IPP”) and dimethylallyl pyrophosphate (“DMAPP”) togeranyl pyrophosphate (“GPP”), farnesyl pyrophosphate (“FPP”), andgeranylgeranyl pyrophosphate (“GGPP”), and the synthesis of variousisoprenoids.

FIG. 3 shows a map of expression plasmid pMBIS-gpps.

FIG. 4 shows a map of expression plasmid pAM408.

FIG. 5 shows a map of expression plasmid pAM424.

FIG. 6 shows a map of expression plasmids pTrc99A-ADS, pTrc99A-FSA,pTrc99A-LLS, pTrc99A-LMS, pTrc99A-GTS, pTrc99A-APS, pTrc99A-BPS,pTrc99A-PHS, pTrc99A-TS, pTrc99A-CS, pTrc99A-SS, and pAM373.

FIGS. 7A-7C are schematics for the construction of plasmidspAM489-pAM498 and for pAM328.

FIG. 8 shows the higher specific activity and increased stability of theEnterococcus faecalis HMGR-CoA reductase (HMGR) compared to theSaccharomyces cerevisiae truncated HMG-CoA reductase (tHMGR).

FIG. 9 shows the relationship between dry cell weight (“DCW”) per literand OD₆₀₀.

FIGS. 10A-10B show the increased volumetric and specificamorpha-4,11-diene productivity of host strains carrying theStaphylococcus aureus HMGR and HMGS genes compared to host strainscarrying the Saccharomyces cerevisiae tHMGR and HMGS genes.

FIGS. 11A-11B show the effect of lower temperature on amorpha-4,11-dieneproductivity of an Escherichia coli host strain.

FIGS. 12A-12D show the effect of reduced glucose levels onamorpha-4,11-diene productivity of an Escherichia coli host strain.

FIGS. 13A-13B show the combined effects of lower temperature and reducedglucose levels on amorpha-4,11-diene productivity of an Escherichia colihost strain.

FIGS. 14A-14E and 15A-15E show the combined effects of lower temperatureand reduced glucose and nitrogen levels on amorpha-4,11-dieneproductivity of an Escherichia coli host strain.

FIG. 16 shows production of amorpha-4,11-diene via the DXP pathway by anEscherichia coli host strain.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Reference is made here to anumber of terms that shall be defined to have the following meanings:

The term “optional” or “optionally” means that the subsequentlydescribed feature or structure may or may not be present, or that thesubsequently described event or circumstance may or may not occur, andthat the description includes instances where a particular feature orstructure is present and instances where the feature or structure isabsent, or instances where the event or circumstance occurs andinstances where the event or circumstance does not occur.

The terms “metabolic pathway” is used herein to refer to a catabolicpathway or an anabolic pathway. Anabolic pathways involve constructing alarger molecule from smaller molecules, a process requiring energy.Catabolic pathways involve breaking down of larger molecules, oftenreleasing energy.

The term “mevalonate pathway” or “MEV pathway” is used herein to referto the biosynthetic pathway that converts acetyl-CoA to IPP. The MEVpathway is illustrated schematically in FIG. 1A.

The term “deoxyxylulose 5-phosphate pathway” or “DXP pathway” is usedherein to refer to the pathway that converts glyceraldehyde-3-phosphateand pyruvate to IPP and DMAPP. The DXP pathway is illustratedschematically in FIG. 1B.

The word “pyrophosphate” is used interchangeably herein with“diphosphate”.

The terms “expression vector” or “vector” refer to a nucleic acid thattransduces, transforms, or infects a host cell, thereby causing the cellto produce nucleic acids and/or proteins other than those that arenative to the cell, or to express nucleic acids and/or proteins in amanner that is not native to the cell.

The term “endogenous” refers to a substance or process that occursnaturally, e.g., in a non-recombinant host cell.

The terms “enzymatic pathway for making isopentenyl pyrophosphate”refers to any pathway capable of producing isopentyl pyrophosphate,including, without limitation, either the mevalonate pathway or the DXPpathway.

The term “nucleic acid” refers to a polymeric form of nucleotides of anylength, either ribonucleotides or deoxynucleotides. Thus, this termincludes, but is not limited to, single-, double-, or multi-stranded DNAor RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprisingpurine and pyrimidine bases or other natural, chemically, orbiochemically modified, non-natural, or derivatized nucleotide bases.

The term “operon” is used to refer to two or more contiguous nucleotidesequences that each encode a gene product such as a RNA or a protein,and the expression of which are coordinately regulated by one or morecontrolling elements (for example, a promoter).

The term “gene product” refers to RNA encoded by DNA (or vice versa) orprotein that is encoded by an RNA or DNA, where a gene will typicallycomprise one or more nucleotide sequences that encode a protein, and mayalso include introns and other non-coding nucleotide sequences.

The term “protein” refers to a polymeric form of amino acids of anylength, which can include coded and non-coded amino acids, chemically orbiochemically modified or derivatized amino acids, and polypeptideshaving modified peptide backbones.

The term “heterologous nucleic acid” as used herein refers to a nucleicacid wherein at least one of the following is true: (a) the nucleic acidis foreign (“exogenous”) to (that is, not naturally found in) a givenhost cell; (b) the nucleic acid comprises a nucleotide sequence that isnaturally found in (that is, is “endogenous to”) a given host cell, butthe nucleotide sequence is produced in an unnatural (for example,greater than expected or greater than naturally found) amount in thecell; (c) the nucleic acid comprises a nucleotide sequence that differsin sequence from an endogenous nucleotide sequence, but the nucleotidesequence encodes the same protein (having the same or substantially thesame amino acid sequence) and is produced in an unnatural (for example,greater than expected or greater than naturally found) amount in thecell; or (d) the nucleic acid comprises two or more nucleotide sequencesthat are not found in the same relationship to each other in nature (forexample, the nucleic acid is recombinant).

A “transgene” refers to a gene that is exogenously introduced into ahost cell. It can comprise an endogenous or exogenous, or heterologousnucleic acid.

The term “recombinant host” (also referred to as a “genetically modifiedhost cell” or “genetically modified host microorganism”) denotes a hostcell that comprises a heterologous nucleic acid of the invention.

The term “exogenous nucleic acid” refers to a nucleic acid that isexogenously introduced into a host cell, and hence is not normally ornaturally found in and/or produced by a given cell in nature.

The term “regulatory element” refers to transcriptional andtranslational control sequences, such as promoters, enhancers,polyadenylation signals, terminators, protein degradation signals, andthe like, that provide for and/or regulate expression of a codingsequence and/or production of an encoded polypeptide in a host cell.

The term “transformation” refers to a permanent or transient geneticchange induced in a cell following introduction of new nucleic acid.Genetic change (“modification”) can be accomplished either byincorporation of the new DNA into the genome of the host cell, or bytransient or stable maintenance of the new DNA as an episomal element.In eukaryotic cells, a permanent genetic change is generally achieved byintroduction of the DNA into the genome of the cell. In prokaryoticcells, a permanent genetic change can be introduced into the chromosomeor via extrachromosomal elements such as plasmids and expressionvectors, which may contain one or more selectable markers to aid intheir maintenance in the recombinant host cell.

The term “operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. For instance, a promoter is operablylinked to a nucleotide sequence if the promoter affects thetranscription or expression of the nucleotide sequence.

The term “host cell” and “host microorganism” are used interchangeablyherein to refer to any archae, bacterial, or eukaryotic living cell intowhich a heterologous nucleic acid can be or has been inserted. The termalso relates to the progeny of the original cell, which may notnecessarily be completely identical in morphology or in genomic or totalDNA complement to the original parent, due to natural, accidental, ordeliberate mutation.

The term “synthetic” as used in reference to nucleic acids means theannealing of chemically synthesized oligonucleotide building blocks toform gene segments, which are then enzymatically assembled to constructthe entire gene. Synthesis of nucleic acids via “chemical means” meansthat the component nucleotides were assembled in vitro.

The term “natural” as applied to a nucleic acid, a cell, or an organism,refers to a nucleic acid, cell, or organism that is found in nature. Forexample, a polypeptide or polynucleotide sequence that is present in anon-pathological (un-diseased) organism that can be isolated from asource in nature and that has not been intentionally modified by a humanin the laboratory is natural.

The term “naturally occurring” as applied to a nucleic acid, an enzyme,a cell, or an organism, refers to a nucleic acid, enzyme, cell, ororganism that is found in nature. For example, a polypeptide orpolynucleotide sequence that is present in an organism that can beisolated from a source in nature and that has not been intentionallymodified by a human in the laboratory is naturally occurring.

The term “biologically active fragment” as applied to a protein,polypeptide or enzyme refers to functional portion(s) of the proteins orpolypeptide or enzyme. Functionally equivalents may have variant aminoacid sequences may arise, e.g., as a consequence of codon redundancy andfunctional equivalency which are known to occur naturally within nucleicacid sequences and the proteins thus encoded. Functionally equivalentproteins or peptides may alternatively be constructed via theapplication of recombinant DNA technology, in which changes in theprotein structure may be engineered, based on considerations of theproperties of the amino acids being exchanged.

The terms “isoprenoid”, “isprenoid compound”, “isoprenoid product”,“terpene”, “terpene compound”, “terpenoid”, and “terpenoid compound” areused interchangeably herein. They refer to compounds that are capable ofbeing derived from IPP.

The singular forms “a,” “and,” and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference to“an expression vector” includes a single expression vector as well as aplurality of expression vectors, and reference to “the host cell”includes reference to one or more host cells, and so forth. It isfurther noted that the claims may be drafted to exclude any optionalelement. As such, this statement is intended to serve as antecedentbasis for use of such exclusive terminology as “solely,” “only” and thelike in connection with the recitation of claim elements, or use of a“negative” limitation.

Unless otherwise indicated, this invention is not limited to particularsequences, expression vectors, enzymes, host microorganisms, orprocesses, as such may vary in accordance with the understanding ofthose of ordinary skill in the arts to which this invention pertains inview of the teaching herein. Terminology used herein is for purposes ofdescribing particular embodiments only and is not intended to belimiting.

Host Cells

Any suitable host cell may be used in the practice of the presentinvention. In one embodiment, the host cell is a genetically modifiedhost microorganism in which nucleic acid molecules have been inserted,deleted or modified (i.e., mutated; e.g., by insertion, deletion,substitution, and/or inversion of nucleotides), to either produce thedesired isoprenoid compound or isoprenoid derivative, or effect anincreased yield of the desired isoprenoid compound or isoprenoidderivative. In another embodiment, the host cell is capable of beinggrown in liquid growth medium. In contrast, a “control cell” is analternative subject or sample used in an experiment for comparisonpurpose, and is typically a parental cell that does not contain themodification(s) made to a corresponding host cell.

Illustrative examples of suitable host cells include any archae,prokaryotic, or eukaryotic cell. Examples of an archae cell include, butare not limited to those belonging to the genera: Aeropyrum,Archaeglobus, Halobacterium, Methanococcus, Methanobacterium,Pyrococcus, Sulfolobus, and Thermoplasma. Illustrative examples ofarchae strains include but are not limited to: Aeropyrum pernix,Archaeoglobus fulgidus, Methanococcus jannaschii, Methanobacteriumthermoautotrophicum, Pyrococcus abyssi, Pyrococcus horikoshii,Thermoplasma acidophilum, Thermoplasma volcanium.

Examples of a procaryotic cell include, but are not limited to thosebelonging to the genera: Agrobacterium, Alicyclobacillus, Anabaena,Anacystis, Arthrobacter, Azobacter, Bacillus, Brevibacterium,Chromatium, Clostridium, Corynebacterium, Enterobacter, Erwinia,Escherichia, Lactobacillus, Lactococcus, Mesorhizobium,Methylobacterium, Microbacterium, Phormidium, Pseudomonas, Rhodobacter,Rhodopseudomonas, Rhodospirillum, Rhodococcus, Salmonella, Scenedesmun,Serratia, Shigella, Staphlococcus, Strepromyces, Synnecoccus, andZymomonas.

Illustrative examples of prokaryotic bacterial strains include but arenot limited to: Bacillus subtilis, Bacillus amyloliquefacines,Brevibacterium ammoniagenes, Brevibacterium immariophilum, Clostridiumbeigerinckii, Enterobacter sakazakii, Escherichia coli, Lactococcuslactis, Mesorhizobium loti, Pseudomonas aeruginosa, Pseudomonasmevalonii, Pseudomonas pudica, Rhodobacter capsulatus, Rhodobactersphaeroides, Rhodospirillum rubrum, Salmonella enterica, Salmonellatyphi, Salmonella typhimurium, Shigella dysenteriae, Shigella flexneri,Shigella sonnei, Staphylococcus aureus, and the like.

In general, if a bacterial host cell is used, a non-pathogenic strain ispreferred. Illustrative examples of non-pathogenic strains include butare not limited to: Bacillus subtilis, Escherichia coli, Lactibacillusacidophilus, Lactobacillus helveticus, Pseudomonas aeruginosa,Pseudomonas mevalonii, Pseudomonas pudita, Rhodobacter sphaeroides,Rodobacter capsulatus, Rhodospirillum rubrum, and the like.

Examples of eukaryotic cells include but are not limited to fungalcells. Examples of fungal cell include, but are not limited to thosebelonging to the genera: Aspergillus, Candida, Chrysosporium,Cryotococcus, Fusarium, Kluyveromyces, Neotyphodium, Neurospora,Penicillium, Pichia, Saccharomyces, Trichoderma and Xanthophyllomyces(formerly Phaffia).

Illustrative examples of eukaryotic strains include but are not limitedto: Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Candidaalbicans, Chrysosporium lucknowense, Fusarium graminearum, Fusariumvenenatum, Kluyveromyces lactis, Neurospora crassa, Pichia angusta,Pichia finlandica, Pichia kodamae, Pichia membranaefaciens, Pichiamethanolica, Pichia opuntiae, Pichia pastoris, Pichia pijperi, Pichiaquercuum, Pichia salictaria, Pichia thermotolerans, Pichia trehalophila,Pichia stipitis, Streptomyces ambofaciens, Streptomyces aureofaciens,Streptomyces aureus, Saccaromyces bayanus, Saccaromyces boulardi,Saccharomyces cerevisiae, Streptomyces fungicidicus, Streptomycesgriseochromogenes, Streptomyces griseus, Streptomyces lividans,Streptomyces olivogriseus, Streptomyces rameus, Streptomycestanashiensis, Streptomyces vinaceus, Trichoderma reesei andXanthophyllomyces dendrorhous (formerly Phaffia rhodozyma).

In general, if a eukaryotic cell is used, a non-pathogenic strain ispreferred. Illustrative examples of non-pathogenic strains include butare not limited to: Fusarium graminearum, Fusarium venenatum, Pichiapastoris, Saccaromyces boulardi, and Saccaromyces cerevisiae.

In addition, certain strains have been designated by the Food and DrugAdministration as

GRAS or Generally Regarded As Safe. These strains include: Bacillussubtilis, Lactibacillus acidophilus, Lactobacillus helveticus, andSaccharomyces cerevisiae.

IPP Pathways

The host cells of the present invention comprise or utilize the MEVpathway, the DXP pathway or both to synthesize IPP and its isomer,DMAPP. In general, eukaryotes other than plants use the MEV isoprenoidpathway exclusively to convert acetyl-CoA to IPP, which is subsequentlyisomerized to DMAPP. Prokaryotes, with some exceptions, use themevalonate-independent or DXP pathway to produce IPP and DMAPPseparately through a branch point. In general, plants use both the MEVand DXP pathways for IPP synthesis.

MEV Pathway

A schematic representation of the MEV pathway is described in FIG. 1A.In general, the pathway comprises six steps.

In the first step, two molecules of acetyl-coenzyme A are enzymaticallycombined to form acetoacetyl-CoA. An enzyme known to catalyze this stepis, for example, acetyl-CoA thiolase (also known as acetyl-CoAacetyltransferase). Illustrative examples of nucleotide sequencesinclude but are not limited to the following GenBank accession numbersand the organism from which the sequences derived: (NC_(—)000913 REGION:2324131 . . . 2325315; Escherichia coli), (D49362; Paracoccusdenitrificans), and (L20428; Saccharomyces cerevisiae).

In the second step of the MEV pathway, acetoacetyl-CoA is enzymaticallycondensed with another molecule of acetyl-CoA to form3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). An enzyme known to catalyzethis step is, for example, HMG-CoA synthase. Illustrative examples ofnucleotide sequences include but are not limited to: (NC_(—)001145.complement 19061.20536; Saccharomyces cerevisiae), (X96617;Saccharomyces cerevisiae), (X83882; Arabidopsis thaliana), (AB037907;Kitasatospora griseola), (BT007302; Homo sapiens), and (NC_(—)002758,Locus tag SAV2546, GeneID 1122571; Staphylococcus aureus).

In the third step, HMG-CoA is enzymatically converted to mevalonate. Anenzyme known to catalyze this step is, for example, HMG-CoA reductase.Illustrative examples of nucleotide sequences include but are notlimited to: (NM 206548; Drosophila melanogaster), (NC_(—)002758, Locustag SAV2545, GeneID 1122570; Staphylococcus aureus), (NM 204485; Gallusgallus), (AB015627; Streptomyces sp. KO 3988), (AF542543; Nicotianaattenuata), (AB037907; Kitasatospora griseola), (AX128213, providing thesequence encoding a truncated HMGR; Saccharomyces cerevisiae), and(NC_(—)001145: complement (115734.118898; Saccharomyces cerevisiae).

In the fourth step, mevalonate is enzymatically phosphorylated to formmevalonate 5-phosphate. An enzyme known to catalyze this step is, forexample, mevalonate kinase. Illustrative examples of nucleotidesequences include but are not limited to: (L77688; Arabidopsisthaliana), and (X55875; Saccharomyces cerevisiae).

In the fifth step, a second phosphate group is enzymatically added tomevalonate 5-phosphate to form mevalonate 5-pyrophosphate. An enzymeknown to catalyze this step is, for example, phosphomevalonate kinase.Illustrative examples of nucleotide sequences include but are notlimited to: (AF429385; Hevea brasiliensis), (NM_(—)006556; Homosapiens), and (NC_(—)001145. complement 712315 . . . 713670;Saccharomyces cerevisiae).

In the sixth step, mevalonate 5-pyrophosphate is enzymatically convertedinto IPP. An enzyme known to catalyze this step is, for example,mevalonate pyrophosphate decarboxylase. Illustrative examples ofnucleotide sequences include but are not limited to: (X97557;Saccharomyces cerevisiae), (AF290095; Enterococcus faecium), and(U49260; Homo sapiens).

If IPP is to be converted to DMAPP, then a seventh step is required. Anenzyme known to catalyze this step is, for example, IPP isomerase.Illustrative examples of nucleotide sequences include but are notlimited to: (NC_(—)000913, 3031087.3031635; Escherichia coli), and(AF082326; Haematococcus pluvialis). If the conversion to DMAPP isrequired, an increased expression of IPP isomerase ensures that theconversion of IPP into DMAPP does not represent a rate-limiting step inthe overall pathway.

DXP Pathway

A schematic representation of the DXP pathway is described in FIG. 1B.In general, the DXP pathway comprises seven steps. In the first step,pyruvate is condensed with D-glyceraldehyde 3-phosphate to make1-deoxy-D-xylulose-5-phosphate. An enzyme known to catalyze this stepis, for example, 1-deoxy-D-xylulose-5-phosphate synthase. Illustrativeexamples of nucleotide sequences include but are not limited to:(AF035440; Escherichia coli), (NC_(—)002947, locus tag PP0527;Pseudomonas putida KT2440), (CP000026, locus tag SPA2301; Salmonellaenterica Paratyphi, see ATCC 9150), (NC_(—)007493, locus tagRSP_(—)0254; Rhodobacter sphaeroides 2.4.1), (NC_(—)005296, locus tagRPA0952; Rhodopseudomonas palustris CGA009), (NC_(—)004556, locus tagPD1293; Xylella fastidiosa Temecula1), and (NC_(—)003076, locus tagAT5G11380; Arabidopsis thaliana).

In the second step, 1-deoxy-D-xylulose-5-phosphate is converted to2C-methyl-D-erythritol-4-phosphate. An enzyme known to catalyze thisstep is, for example, 1-deoxy-D-xylulose-5-phosphate reductoisomerase.Illustrative examples of nucleotide sequences include but are notlimited to: (AB013300; Escherichia coli), (AF148852; Arabidopsisthaliana), (NC_(—)002947, locus tag PP1597; Pseudomonas putida KT2440),(AL939124, locus tag SCO5694; Streptomyces coelicolor A3(2)),(NC_(—)007493, locus tag RSP_(—)2709; Rhodobacter sphaeroides 2.4.1),and (NC_(—)007492, locus tag Pfl_(—)1107; Pseudomonas fluorescensPfO-1).

In the third step, 2C-methyl-D-erythritol-4-phosphate is converted to4-diphosphocytidyl-2C-methyl-D-erythritol. An enzyme known to catalyzethis step is, for example, 4-diphosphocytidyl-2C-methyl-D-erythritolsynthase. Illustrative examples of nucleotide sequences include but arenot limited to: (AF230736; Escherichia coli), (NC_(—)007493, locus_tagRSP_(—)2835; Rhodobacter sphaeroides 2.4.1), (NC_(—)003071, locus_tagAT2G02500; Arabidopsis thaliana), and (NC_(—)002947, locus_tag PP1614;Pseudomonas putida KT2440).

In the fourth step, 4-diphosphocytidyl-2C-methyl-D-erythritol isconverted to 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate. Anenzyme known to catalyze this step is, for example,4-diphosphocytidyl-2C-methyl-D-erythritol kinase. Illustrative examplesof nucleotide sequences include but are not limited to: (AF216300;Escherichia coli) and (NC_(—)007493, locus_tag RSP_(—)1779; Rhodobactersphaeroides 2.4.1).

In the fifth step, 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphateis converted to 2C-methyl-D-erythritol 2,4-cyclodiphosphate. An enzymeknown to catalyze this step is, for example, 2C-methyl-D-erythritol2,4-cyclodiphosphate synthase. Illustrative examples of nucleotidesequences include but are not limited to: (AF230738; Escherichia coli),(NC_(—)007493, locus_tag RSP_(—)6071; Rhodobacter sphaeroides 2.4.1),and (NC_(—)002947, locus_tag PP1618; Pseudomonas putida KT2440).

In the sixth step, 2C-methyl-D-erythritol 2,4-cyclodiphosphate isconverted to 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate. An enzymeknown to catalyze this step is, for example,1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase. Illustrativeexamples of nucleotide sequences include but are not limited to:(AY033515; Escherichia coli), (NC_(—)002947, locus_tag PP0853;Pseudomonas putida KT2440), and (NC_(—)007493, locus_tag RSP_(—)2982;Rhodobacter sphaeroides 2.4.1).

In the seventh step, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate isconverted into either IPP or its isomer, DMAPP. An enzyme known tocatalyze this step is, for example, isopentyl/dimethylallyl diphosphatesynthase. Illustrative examples of nucleotide sequences include but arenot limited to: (AY062212; Escherichia coli) and (NC_(—)002947,locus_tag PP0606; Pseudomonas putida KT2440).

In some embodiments, “cross talk” (or interference) between the hostcell's own metabolic processes and those processes involved with theproduction of IPP as provided by the present invention are minimized oreliminated entirely. For example, cross talk is minimized or eliminatedentirely when the host microorganism relies exclusively on the DXPpathway for synthesizing IPP, and a MEV pathway is introduced to provideadditional IPP. Such a host organisms would not be equipped to alter theexpression of the MEV pathway enzymes or process the intermediatesassociated with the MEV pathway. Organisms that rely exclusively orpredominately on the DXP pathway include, for example, Escherichia coli.

In some embodiments, the host cell produces IPP via the MEV pathway,either exclusively or in combination with the DXP pathway. In otherembodiments, a host's DXP pathway is functionally disabled so that thehost cell produces IPP exclusively through a heterologously introducedMEV pathway. The DXP pathway can be functionally disabled by disablinggene expression or inactivating the function of one or more of the DXPpathway enzymes.

C₅ Compounds

C₅ compounds of the invention generally are derived from IPP or DMAPP.These compounds are also known as hemiterpenes because they are derivedfrom a single isoprene unit (IPP or DMAPP).

Isoprene

Isoprene, whose structure is

is found in many plants. Isoprene is made from IPP by isoprene synthase.Illustrative examples of suitable nucleotide sequences include but arenot limited to: (AB 198190; Populus alba) and (AJ294819; Polulusalba×Polulus tremula).C₁₀ Compounds

C₁₀ compounds of the invention generally derived from geranylpyrophosphate (GPP) which is made by the condensation of IPP with DMAPP.An enzyme known to catalyze this step is, for example, geranylpyrophosphate synthase. These C₁₀ compounds are also known asmonoterpenes because they are derived from two isoprene units.

FIG. 2 shows schematically how IPP and DMAPP can produce GPP, which canbe further processed to a monoterpene.

Illustrative examples of nucleotide sequences for geranyl pyrophosphatesynthase include but are not limited to: (AF513111; Abies grandis),(AF513112; Abies grandis), (AF513113; Abies grandis), (AY534686;Antirrhinum majus), (AY534687; Antirrhinum majus), (Y17376; Arabidopsisthaliana), (AE016877, Locus AP11092; Bacillus cereus; ATCC 14579),(AJ243739; Citrus sinensis), (AY534745; Clarkia breweri), (AY953508; Ipspini), (DQ286930; Lycopersicon esculentum), (AF182828; Mentha×piperita),(AF182827; Mentha×piperita), (MPI249453; Mentha×piperita), (PZE431697,Locus CAD24425; Paracoccus zeaxanthinifaciens), (AY866498; Picrorhizakurrooa), (AY351862; Vitis vinifera), and (AF203881, Locus AAF12843;Zymomonas mobilis).

GPP is then subsequently converted to a variety of C₁₀ compounds.Illustrative examples of C₁₀ compounds include but are not limited:

Carene

Carene, whose structure is

is found in the resin of many trees, particularly pine trees. Carene ismade from GPP from carene synthase. Illustrative examples of suitablenucleotide sequences include but are not limited to: (AF461460, REGION43.1926; Picea abies) and (AF527416, REGION: 78.1871; Salviastenophylla).

Geraniol

Geraniol (also known as rhodnol), whose structure is

is the main component of oil-of-rose and palmarosa oil. It also occursin geranium, lemon, and citronella. Geraniol is made from GPP bygeraniol synthase. Illustrative examples of suitable nucleotidesequences include but are not limited to: (AJ457070; Cinnamomumtenuipilum), (AY362553; Ocimum basilicum), (DQ234300; Perilla frutescensstrain 1864), (DQ234299; Perilla citriodora strain 1861), (DQ234298;Perilla citriodora strain 4935), and (DQ088667; Perilla citriodora)

Linalool

Linalool, whose structure is

is found in many flowers and spice plants such as coriander seeds.Linalool is made from GPP by linalool synthase. Illustrative examples ofa suitable nucleotide sequence include but are not limited to:(AF497485; Arabidopsis thaliana), (AC002294, Locus AAB71482; Arabidopsisthaliana), (AY059757; Arabidopsis thaliana), (NM_(—)104793; Arabidopsisthaliana), (AF154124; Artemisia annua), (AF067603; Clarkia breweri),(AF067602; Clarkia concinna), (AF067601; Clarkia breweri), (U58314;Clarkia breweri), (AY840091; Lycopersicon esculentum), (DQ263741;Lavandula angustifolia), (AY083653; Mentha citrate), (AY693647; Ocimumbasilicum), (XM_(—)463918; Oryza sativa), (AP004078, Locus BAD07605;Oryza sativa), (XM_(—)463918, Locus XP_(—)463918; Oryza sativa),(AY917193; Perilla citriodora), (AF271259; Perilla frutescens),(AY473623; Picea abies), (DQ195274; Picea sitchensis), and (AF444798;Perilla frutescens var. crispa cultivar No. 79).

Limonene

Limonene, whose structure is

is found in the rind of citrus fruits and peppermint. Limonene is madefrom GPP by limonene synthase. Illustrative examples of suitablenucleotide sequences include but are not limited to: (+)-limonenesynthases (AF514287, REGION: 47.1867; Citrus limon) and (AY055214,REGION: 48.1889; Agastache rugosa) and (−)-limonene synthases (DQ195275,REGION: 1.1905; Picea sitchensis), (AF006193, REGION: 73.1986; Abiesgrandis), and (MHC4SLSP, REGION: 29.1828; Mentha spicata).

Myrcene

Myrcene, whose structure is

is found in the essential oil in many plants including bay, verbena, andmyrcia from which it gets its name. Myrcene is made from GPP by myrcenesynthase. Illustrative examples of suitable nucleotide sequences includebut are not limited to: (U87908; Abies grandis), (AY195609; Antirrhinummajus), (AY195608; Antirrhinum majus), (NM 127982; Arabidopsis thalianaTPS10), (NM_(—)113485; Arabidopsis thaliana ATTPS-CIN), (NM_(—)113483;Arabidopsis thaliana ATTPS-CIN), (AF271259; Perilla frutescens),(AY473626; Picea abies), (AF369919; Picea abies), and (AJ304839; Quercusilex).

Ocimene

α- and β-Ocimene, whose structures are

respectively, are found in a variety of plants and fruits includingOcimum basilicum and is made from GPP by ocimene synthase. Illustrativeexamples of suitable nucleotide sequences include but are not limitedto: (AY195607; Antirrhinum majus), (AY195609; Antirrhinum majus),(AY195608; Antirrhinum majus), (AK221024; Arabidopsis thaliana),(NM_(—)113485; Arabidopsis thaliana ATTPS-CIN), (NM_(—)113483;Arabidopsis thaliana ATTPS-CIN), (NM_(—)117775; Arabidopsis thalianaATTPS03), (NM_(—)001036574; Arabidopsis thaliana ATTPS03), (NM 127982;Arabidopsis thaliana TPS10), (AB110642; Citrus unshiu CitMTSL4), and(AY575970; Lotus corniculatus var. japonicus).

α-Pinene

α-Pinene, whose structure is

is found in pine trees and eucalyptus. α-Pinene is made from GPP byα-pinene synthase. Illustrative examples of suitable nucleotidesequences include but are not limited to: (+) α-pinene synthase(AF543530, REGION: 1.1887; Pinus taeda), (−)α-pinene synthase (AF543527,REGION: 32.1921; Pinus taeda), and (+)/(−)α-pinene synthase (AGU87909,REGION: 6111892; Abies grandis).

β-Pinene

β-Pinene, whose structure is

is found in pine trees, rosemary, parsley, dill, basil, and rose.β-Pinene is made from GPP by β-pinene synthase. Illustrative examples ofsuitable nucleotide sequences include but are not limited to: (−)β-pinene synthases (AF276072, REGION: 1.1749; Artemisia annua) and(AF514288, REGION: 26.1834; Citrus limon).

Sabinene

Sabinene, whose structure is

is found in black pepper, carrot seed, sage, and tea trees. Sabinene ismade from GPP by sabinene synthase. An illustrative example of asuitable nucleotide sequence includes but is not limited to AF051901,REGION: 26.1798 from Salvia officinalis.

γ-Terpinene

γ-Terpinene, whose structure is

is a constituent of the essential oil from citrus fruits. Biochemically,γ-terpinene is made from GPP by a γ-terpinene synthase. Illustrativeexamples of suitable nucleotide sequences include: (AF514286, REGION:30.1832 from Citrus limon) and (AB110640, REGION 1.1803 from Citrusunshiu).

Terpinolene

Terpinolene, whose structure is

is found in black currant, cypress, guava, lychee, papaya, pine, andtea. Terpinolene is made from GPP by terpinolene synthase. Anillustrative example of a suitable nucleotide sequence includes but isnot limited to AY906866, REGION: 10.1887 from Pseudotsuga menziesii.C₁₅ Compounds

C₁₅ compounds of the invention generally derive from farnesylpyrophosphate (FPP) which is made by the condensation of two moleculesof IPP with one molecule of DMAPP. An enzyme known to catalyze this stepis, for example, farnesyl pyrophosphate synthase. These C₁₅ compoundsare also known as sesquiterpenes because they are derived from threeisoprene units.

FIG. 2 shows schematically how IPP and DMAPP can be combined to produceFPP, which can be further processed to a sesquiterpene.

Illustrative examples of nucleotide sequences for farnesyl pyrophosphatesynthase include but are not limited to: (ATU80605; Arabidopsisthaliana), (ATHFPS2R; Arabidopsis thaliana), (AAU36376; Artemisiaannua), (AF461050; Bos taurus), (D00694; Escherichia coli K-12),(AE009951, Locus AAL95523; Fusobacterium nucleatum subsp. nucleatum ATCC25586), (GFFPPSGEN; Gibberella fujikuroi), (CP000009, Locus AAW60034;Gluconobacter oxydans 621H), (AF019892; Helianthus annuus), (HUMFAPS;Homo sapiens), (KLPFPSQCR; Kluyveromyces lactis), (LAU15777; Lupinusalbus), (LAU20771; Lupinus albus), (AF309508; Mus musculus), (NCFPPSGEN;Neurospora crassa), (PAFPS1; Parthenium argentatum), (PAFPS2; Partheniumargentatum), (RATFAPS; Rattus norvegicus), (YSCFPP; Saccharomycescerevisiae), (D89104; Schizosaccharomyces pombe), (CP000003, LocusAAT87386; Streptococcus pyogenes), (CP000017, Locus AAZ51849;Streptococcus pyogenes), (NC_(—)008022, Locus YP_(—)598856;Streptococcus pyogenes MGAS10270), (NC_(—)008023, Locus YP_(—)600845;Streptococcus pyogenes MGAS2096), (NC_(—)008024, Locus YP_(—)602832;Streptococcus pyogenes MGAS10750), and (MZEFPS; Zea mays).

Alternatively, FPP can also be made by adding IPP to GPP. Illustrativeexamples of nucleotide sequences encoding for an enzyme capable of thisreaction include but are not limited to: (AE000657, Locus AAC06913;Aquifex aeolicus VF5), (NM 202836; Arabidopsis thaliana), (D84432, LocusBAA12575; Bacillus subtilis), (U12678, Locus AAC28894; Bradyrhizobiumjaponicum USDA 110), (BACFDPS; Geobacillus stearothermophilus),(NC_(—)002940, Locus NP_(—)873754; Haemophilus ducreyi 35000HP),(L42023, Locus AAC23087; Haemophilus influenzae Rd KW20), (J05262; Homosapiens), (YP_(—)395294; Lactobacillus sakei subsp. sakei 23K),(NC_(—)005823, Locus YP_(—)000273; Leptospira interrogans serovarCopenhageni str. Fiocruz L1-130), (AB003187; Micrococcus luteus),(NC_(—)002946, Locus YP_(—)208768; Neisseria gonorrhoeae FA 1090),(U00090, Locus AAB91752; Rhizobium sp. NGR234), (J05091; Saccharomycescerevisae), (CP000031, Locus AAV93568; Silicibacter pomeroyi DSS-3),(AE008481, Locus AAK99890; Streptococcus pneumoniae R6), and(NC_(—)004556, Locus NP 779706; Xylella fastidiosa Temecula1).

FPP is then subsequently converted to a variety of C₁₅ compounds.Illustrative examples of C₁₅ compounds include but are not limited to:

Amorphadiene

Amorphadiene, whose structure is

is a precursor to artemisinin which is made by Artemisia anna.Amorphadiene is made from FPP by amorphadiene synthase. An illustrativeexample of a suitable nucleotide sequence is SEQ ID NO. 37 of U.S. Pat.No. 7,192,751.

α-Farnesene

α-Farnesene, whose structure is

is found in various biological sources including but not limited to theDufour's gland in ants and in the coating of apple and pear peels.α-Farnesene is made from FPP by α-farnesene synthase. Illustrativeexamples of suitable nucleotide sequences include but are not limited toDQ309034 from Pyrus communis cultivar d'Anjou (pear; gene name AFS1) andAY182241 from Malus domestica (apple; gene AFS1). Pechouus et al.,Planta 219(1):84-94 (2004).

β-Farnesene

β-Farnesene, whose structure is

is found in various biological sources including but not limited toaphids and essential oils such as from peppermint. In some plants suchas wild potato, β-farnesene is synthesized as a natural insectrepellent. β-Farnesene is made from FPP by β-farnesene synthase.Illustrative examples of suitable nucleotide sequences include but isnot limited to GenBank accession number AF024615 from Mentha×piperita(peppermint; gene Tspa11), and AY835398 from Artemisia annua. Picaud etal., Phytochemistry 66(9): 961-967 (2005).

Farnesol

Farnesol, whose structure is

is found in various biological sources including insects and essentialoils such as from cintronella, neroli, cyclamen, lemon grass, tuberose,and rose. Farnesol is made from FPP by a hydroxylase such as farnesolsynthase. Illustrative examples of suitable nucleotide sequences includebut are not limited to GenBank accession number AF529266 from Zea maysand YDR481C from Saccharomyces cerevisiae (gene Pho8). Song, L., AppliedBiochemistry and Biotechnology 128:149-158 (2006).

Nerolidol

Nerolidol, whose structure is

is also known as peruviol, and is found in various biological sourcesincluding as essential oils such as from neroli, ginger, jasmine,lavender, tea tree, and lemon grass. Nerolidol is made from FPP by ahydroxylase such as nerolidol synthase. An illustrative example of asuitable nucleotide sequence includes but is not limited to AF529266from Zea mays (maize; gene tps1).

Patchoulol

Patchoulol, whose structure is

is also known as patchouli alcohol and is a constituent of the essentialoil of Pogostemon patchouli. Patchouliol is made from FPP by patchouliolsynthase. An illustrative example of a suitable nucleotide sequenceincludes but is not limited to AY508730 REGION: 1.1659 from Pogostemoncablin.

Valencene

Valencene, whose structure is

is one of the main chemical components of the smell and flavour oforanges and is found in orange peels. Valencene is made from FPP bynootkatone synthase. Illustrative examples of a suitable nucleotidesequence includes but is not limited to AF441124 REGION: 1.1647 fromCitrus sinensis and AY917195 REGION: 1.1653 from Perilla frutescens.C₂₀ Compounds

C₂₀ compounds of the invention generally derived from geranylgeraniolpyrophosphate (GGPP) which is made by the condensation of threemolecules of IPP with one molecule of DMAPP. An enzyme known to catalyzethis step is, for example, geranylgeranyl pyrophosphate synthase. TheseC₂₀ compounds are also known as diterpenes because they are derived fromfour isoprene units.

FIG. 2 shows schematically how IPP and DMAPP can be combined to produceGGPP, which can be further processed to a diterpene, or can be furtherprocessed to produce a carotenoid.

Illustrative examples of nucleotide sequences for geranylgeranylpyrophosphate synthase include but are not limited to: (ATHGERPYRS;Arabidopsis thaliana), (BT005328; Arabidopsis thaliana), (NM_(—)119845;Arabidopsis thaliana), (NZ_AAJM01000380, Locus ZP_(—)00743052; Bacillusthuringiensis serovar israelensis, ATCC 35646 sq1563), (CRGGPPS;Catharanthus roseus), (NZ_AABF02000074, Locus ZP_(—)00144509;Fusobacterium nucleatum subsp. vincentii, ATCC 49256), (GFGGPPSGN;Gibberella fujikuroi), (AY371321; Ginkgo biloba), (AB055496; Heveabrasiliensis), (AB017971; Homo sapiens), (MCI276129; Mucorcircinelloides f. lusitanicus), (AB016044; Mus musculus), (AABX01000298,Locus NCU01427; Neurospora crassa), (NCU20940; Neurospora crassa),(NZ_AAKL01000008, Locus ZP_(—)00943566; Ralstonia solanacearum UW551),(AB118238; Rattus norvegicus), (SCU31632; Saccharomyces cerevisiae),(AB016095; Synechococcus elongates), (SAGGPS; Sinapis alba), (SSOGDS;Sulfolobus acidocaldarius), (NC_(—)007759, Locus YP_(—)461832;Syntrophus aciditrophicus SB), and (NC_(—)006840, Locus YP_(—)204095;Vibrio fischeri ES 114).

Alternatively, GGPP can also be made by adding IPP to FPP. Illustrativeexamples of nucleotide sequences encoding an enzyme capable of thisreaction include but are not limited to: (NM 112315; Arabidopsisthaliana), (ERWCRTE; Pantoea agglomerans), (D90087, Locus BAA14124;Pantoea ananatis), (X52291, Locus CAA36538; Rhodobacter capsulatus),(AF195122, Locus AAF24294; Rhodobacter sphaeroides), and (NC_(—)004350,Locus NP_(—)721015; Streptococcus mutans UA159).

GGPP is then subsequently converted to a variety of C₂₀ isoprenoids.Illustrative examples of C₂₀ compounds include but are not limited to:

Geranylgeraniol

Geranylgeraniol, whose structure is

is a constituent of wood oil from Cedrela toona and of linseed oil.Geranylgeraniol can be made by e.g., adding to the expression constructsa phosphatase gene after the gene for a GGPP synthase.

Abietadiene

Abietadiene encompasses the following isomers:

and is found in trees such as Abies grandis. Abietadiene is made byabietadiene synthase. An illustrative example of a suitable nucleotidesequence includes but are not limited to: (U50768; Abies grandis) and(AY473621; Picea abies).C₂₀₊ Compounds

C₂₀₊ compounds are also within the scope of the present invention.Illustrative examples of such compounds include sesterterpenes (C₂₅compound made from five isoprene units), triterpenes (C₃₀ compounds madefrom six isoprene units), and tetraterpenes (C₄₀ compound made fromeight isoprene units). These compounds are made by using similar methodsdescribed herein and substituting or adding nucleotide sequences for theappropriate synthase(s).

Engineering Pathways

The present invention utilizes an engineered MEV and/or DXP pathway toeffect the high-level production of isoprenoids in a host cell. Thepathway is typically engineered via recombinant DNA technology byexpressing heterologous sequences encoding enzymes in at least one ofthese pathways.

The subject nucleotide acids can be expressed by a single or multiplevectors. For example, a single expression vector can comprise at leasttwo, three, four, five, or all of the heterologous sequences encodingthe entire MEV or DXP pathway enzymes. While the choice of single ormultiple vectors may depend on the size of the heterologous sequencesand the capacity of the vectors, it will largely dependent on theoverall yield of a given isoprenoid that the vector is able to providewhen expressed in a selected host cell. The subject vectors can stayreplicable episomally, or as an integral part of the host cell genome.Typically, the latter is preferred for a sustained propagation of thehost cell.

In certain host cells, the one or more heterologous sequences encodingthe MEV or DXP pathway enzymes may be controlled by one or more operons.In some instances, a two or three operon system provides a higher yieldof an isoprenoid over a single operon system.

Where desired, the subject nucleic acid sequences can be modified toreflect the codon preference of a selected host cell to effect a higherexpression of such sequences in a host cell. For example, the subjectnucleotide sequences will in some embodiments be modified for yeastcodon preference. See, e.g., Bennetzen and Hall (1982) J: Biol. Chem.257(6): 3026-3031. As another non-limiting example, the nucleotidesequences will in other embodiments be modified for E. coli codonpreference. See, e.g., Gouy and Gautier (1982) Nucleic Acids Res.10(22):7055-7074; Eyre-Walker (1996) Mol. Biol. Evol. 13(6):864-872. Seealso Nakamura et al. (2000) Nucleic Acids Res. 28(1):292. Codon usagetables for many organisms are available, which can be used as areference in designing sequences of the present invention. The use ofprevalent codons of a given host microorganism generally increases thelikelihood of translation, and hence the expression level of the desiredsequences.

Preparation of the subject nucleic acids can be carried out by a varietyof routine recombinant techniques and synthetic procedures. Briefly, thesubject nucleic acids can be prepared genomic DNA fragments, cDNAs, andRNAs, all of which can be extracted directly from a cell orrecombinantly produced by various amplification processes including butnot limited to PCR and rt-PCR.

Direct chemical synthesis of nucleic acids typically involves sequentialaddition of 3′-blocked and 5′-blocked nucleotide monomers to theterminal 5′-hydroxyl group of a growing nucleotide polymer chain,wherein each addition is effected by nucleophilic attack of the terminal5′-hydroxyl group of the growing chain on the 3′-position of the addedmonomer, which is typically a phosphorus derivative, such as aphosphotriester, phosphoramidite, or the like. Such methodology is knownto those of ordinary skill in the art and is described in the pertinenttexts and literature (for example, Matteuci et al. (1980) Tet. Lett.521:719; U.S. Pat. No. 4,500,707 to Caruthers et al.; and U.S. Pat. Nos.5,436,327 and 5,700,637 to Southern et al.).

The level of transcription of a nucleic acid in a host microorganism canbe increased in a number of ways. For example, this can be achieved byincreasing the copy number of the nucleotide sequence encoding theenzyme (e.g., by using a higher copy number expression vector comprisinga nucleotide sequence encoding the enzyme, or by introducing additionalcopies of a nucleotide sequence encoding the enzyme into the genome ofthe host microorganism, for example, by recA-mediated recombination, useof “suicide” vectors, recombination using lambda phage recombinase,and/or insertion via a transposon or transposable element). In addition,it can be carried out by changing the order of the coding regions on thepolycistronic mRNA of an operon or breaking up an operon into individualgenes, each with its own control elements, or increasing the strength ofthe promoter (transcription initiation or transcription controlsequence) to which the enzyme coding region is operably linked (forexample, using a consensus arabinose- or lactose-inducible promoter inan Escherichia coli host microorganism in place of a modifiedlactose-inducible promoter, such as the one found in pBluescript and thepBBR1MCS plasmids), or using an inducible promoter and inducing theinducible-promoter by adding a chemical to a growth medium. The level oftranslation of a nucleotide sequence in a host microorganism can beincreased in a number of ways, including, but not limited to, increasingthe stability of the mRNA, modifying the sequence of the ribosomebinding site, modifying the distance or sequence between the ribosomebinding site and the start codon of the enzyme coding sequence,modifying the entire intercistronic region located “upstream of” oradjacent to the 5′ side of the start codon of the enzyme coding region,stabilizing the 3′-end of the mRNA transcript using hairpins andspecialized sequences, modifying the codon usage of enzyme, alteringexpression of rare codon tRNAs used in the biosynthesis of the enzyme,and/or increasing the stability of the enzyme, as, for example, viamutation of its coding sequence. Determination of preferred codons andrare codon tRNAs can be based on a sequence analysis of genes derivedfrom the host microorganism.

The activity of a MEV, DXP, or prenyltransferase in a host can bealtered in a number of ways, including, but not limited to, expressing amodified form of the enzyme that exhibits increased solubility in thehost cell, expressing an altered form of the enzyme that lacks a domainthrough which the activity of the enzyme is inhibited, expressing amodified form of the enzyme that has a higher Kcat or a lower Km for thesubstrate, or expressing an altered form of the enzyme that is notaffected by feed-back or feed-forward regulation by another molecule inthe pathway. Such variant enzymes can also be isolated through randommutagenesis of a broader specificity enzyme, as described below, and anucleotide sequence encoding such variant enzyme can be expressed froman expression vector or from a recombinant gene integrated into thegenome of a host microorganism.

The subject vector can be constructed to yield a desired level of copynumbers of the encoded enzyme. In some embodiments, the subject vectorsyield at least 10, between 10 to 20, between 20-50, between 50-100, oreven higher than 100 copies of the HMG-CoA reductase, mevalonate kinase,or both. Low copy number plasmids generally provide fewer than about 20plasmid copies per cell; medium copy number plasmids generally providefrom about 20 plasmid copies per cell to about 50 plasmid copies percell, or from about 20 plasmid copies per cell to about 80 plasmidcopies per cell; and high copy number plasmids generally provide fromabout 80 plasmid copies per cell to about 200 plasmid copies per cell,or more.

Suitable low copy expression vectors for Escherichia coli include, butare not limited to, pACYC184, pBeloBac11, pBR332, pBAD33, pBBR1MCS andits derivatives, pSC101, SuperCos (cosmid), and pWE15 (cosmid). Suitablemedium copy expression vectors for Escherichia coli include, but are notlimited to pTrc99A, pBAD24, and vectors containing a ColE1 origin ofreplication and its derivatives. Suitable high copy number expressionvectors for Escherichia coli include, but are not limited to, pUC,pBluescript, pGEM, and pTZ vectors. Suitable low-copy (centromeric)expression vectors for yeast include, but are not limited to, pRS415 andpRS416 (Sikorski & Hieter (1989) Genetics 122:19-27). Suitable high-copy2 micron expression vectors in yeast include, but are not limited to,pRS425 and pRS426 (Christainson et al. (1992) Gene 110:119-122).Alternative 2 micron expression vectors include non-selectable variantsof the 2 micron vector (Bruschi & Ludwig (1988) Curr. Genet. 15:83-90)or intact 2 micron plasmids bearing an expression cassette (asexemplified in U.S. Pat. Appl. 20050084972) or 2 micron plasmids bearinga defective selection marker such as LEU2d (Erhanrt et al. (1983) J.Bacteriol. 156 (2): 625-635) or URA3d (Okkels (1996) Annals of the NewYork Academy of Sciences 782(1): 202-207).

Regulatory elements include, for example, promoters and operators canalso be engineered to increase the metabolic flux of the MEV or DXPpathways by increasing the expression of one or more genes that play asignificant role in determining the overall yield of an isoprenoidproduced. A promoter is a sequence of nucleotides that initiates andcontrols the transcription of a nucleic acid sequence by an RNApolymerase enzyme. An operator is a sequence of nucleotides adjacent tothe promoter that functions to control transcription of the desirednucleic acid sequence. The operator contains a protein-binding domainwhere a specific repressor protein can bind. In the absence of asuitable repressor protein, transcription initiates through thepromoter. In the presence of a suitable repressor protein, the repressorprotein binds to the operator and thereby inhibits transcription fromthe promoter. Promotors and operators are also referred to astranscriptional regulators.

In some embodiments of the present invention, promoters used inexpression vectors are inducible. In other embodiments, the promotersused in expression vectors are constitutive. In some embodiments, one ormore nucleic acid sequences are operably linked to an induciblepromoter, and one or more other nucleic acid sequences are operablylinked to a constitutive promoter.

Non-limiting examples of suitable promoters for use in prokaryotic hostcells include a bacteriophage T7 RNA polymerase promoter; a trppromoter; a lac operon promoter; a hybrid promoter, for example, alac/tac hybrid promoter, a tac/trc hybrid promoter, a trp/lac promoter,a T7/lac promoter; a trc promoter; a tac promoter, and the like; anaraBAD promoter; in vivo regulated promoters, such as an ssaG promoteror a related promoter (see, for example, U.S. Patent Publication No.20040131637), a pagC promoter (Pulkkinen and Miller, J. Bacteriol.(1991) 173(1):86-93; Alpuche-Aranda et al. (1992) Proc. Natl. Acad. Sci.USA. 89(21):10079-83), a nirB promoter (Harborne et al. (1992) Mol.Micro. 6:2805-2813), and the like (see, for example, Dunstan et al.(1999) Infect. Immun. 67:5133-5141; McKelvie et al. (2004) Vaccine22:3243-3255; and Chatfield et al. (1992) Biotechnol. 10:888-892); asigma70 promoter, for example, a consensus sigma70 promoter (see, forexample, GenBank Accession Nos. AX798980, AX798961, and AX798183); astationary phase promoter, for example, a dps promoter, an spy promoter,and the like; a promoter derived from the pathogenicity island SPI-2(see, for example, WO96/17951); an actA promoter (see, for example,Shetron-Rama et al. (2002) Infect. Immun. 70:1087-1096); an rpsMpromoter (see, for example, Valdivia and Falkow (1996) Mol. Microbiol.22:367 378); a tet promoter (see, for example, Hillen et al. (1989) InSaenger W. and Heinemann U. (eds) Topics in Molecular and StructuralBiology, Protein-Nucleic Acid Interaction. Macmillan, London, UK, Vol.10, pp. 143-162); an SP6 promoter (see, for example, Melton et al.(1984) Nucl. Acids Res. 12:7035-7056); and the like.

In some embodiment, the total activity of a heterologous MEV or DXPenzyme that plays a larger role in the overall yield of an isoprenoidrelative to other enzymes in the respective pathways is increased byexpressing the enzyme from a strong promoter. Suitable strong promotersfor Escherichia coli include, but are not limited to Trc, Tac, T5, T7,and P_(Lambda). In another embodiment of the present invention, thetotal activity of the one or more MEV pathway enzymes in a host isincreased by expressing the enzyme from a strong promoter on a high copynumber plasmid. Suitable examples, for Escherichia coli include, but arenot limited to using Trc, Tac, T5, T7, and P_(Lambda) promoters withpBAD24, pBAD18, pGEM, pBluescript, pUC, and pTZ vectors.

Non-limiting examples of suitable promoters for use in eukaryotic hostcells include, but are not limited to, a CMV immediate early promoter,an HSV thymidine kinase promoter, an early or late SV40 promoter, LTRsfrom retroviruses, and a mouse metallothionein-I promoter.

Non-limiting examples of suitable constitutive promoters for use inprokaryotic host cells include a sigma70 promoter (for example, aconsensus sigma70 promoter). Non-limiting examples of suitable induciblepromoters for use in bacterial host cells include the pL ofbacteriophage λ; Plac; Ptrp; Ptac (Ptrp-lac hybrid promoter); anisopropyl-beta-D44 thiogalactopyranoside (IPTG)-inducible promoter, forexample, a lacZ promoter; a tetracycline inducible promoter; anarabinose inducible promoter, for example, PBAD (see, for example,Guzman et al. (1995) J. Bacteriol. 177:4121-4130); a xylose-induciblepromoter, for example, Pxyl (see, for example, Kim et al. (1996) Gene181:71-76); a GAL1 promoter; a tryptophan promoter; a lac promoter; analcohol-inducible promoter, for example, a methanol-inducible promoter,an ethanol-inducible promoter; a raffinose-inducible promoter; aheat-inducible promoter, for example, heat inducible lambda PL promoter;a promoter controlled by a heat-sensitive repressor (for example,CI857-repressed lambda-based expression vectors; see, for example,Hoffmann et al. (1999) FEMS Microbiol Lett. 177(2):327-34); and thelike.

Non-limiting examples of suitable constitutive promoters for use inyeast host cells include an ADH1, an ADH2, a PGK, or a LEU2 promoter.Non-limiting examples of suitable inducible promoters for use in yeasthost cells include, but are not limited to, a divergentgalactose-inducible promoter such as a GAL 1 or a GAL 10 promoter (Westat al. (1984) Mol. Cell. Biol. 4(11):2467-2478), or a CUP1 promoter.Where desired, the subject vector comprise a promoter that is strongerthan a native E. Coli Lac promoter.

Non-limiting examples of operators for use in bacterial host cellsinclude a lactose promoter operator (Lad repressor protein changesconformation when contacted with lactose, thereby preventing the Ladrepressor protein from binding to the operator), a tryptophan promoteroperator (when complexed with tryptophan, TrpR repressor protein has aconformation that binds the operator; in the absence of tryptophan, theTrpR repressor protein has a conformation that does not bind to theoperator), and a tac promoter operator (see, for example, deBoer et al.(1983) Proc. Natl. Acad. Sci. U.S.A. 80:21-25).

The genes in the expression vector typically will also encode a ribosomebinding site to direct translation (that is, synthesis) of any encodedmRNA gene product. For suitable ribosome binding sites for use inEscherichia coli, see Shine et al. (1975) Nature 254:34, and Steitz, inBiological Regulation and Development: Gene Expression (ed. R. F.Goldberger), vol. 1, p. 349, 1979, Plenum Publishing, N.Y. Insertion ofthe ribosome binding site encoding nucleotide sequence 5′-AAAACA-3′upstream of a coding sequence facilitates efficient translation in ayeast host microorganism (Looman et al. (1993) Nuc. Ac. Res.21:4268-4271; Yun et. al. (1996) Mol. Microbiol. 19:1225-1239).

Other regulatory elements that may be used in an expression vectorinclude transcription enhancer elements and transcription terminators.See, for example, Bitter et al. (1987) Methods in Enzymology,153:516-544.

An expression vector may be suitable for use in particular types of hostmicroorganisms and not others. One of ordinary skill in the art,however, can readily determine through routine experimentation whether aparticular expression vector is suited for a given host microorganism.For example, the expression vector can be introduced into the hostorganism, which is then monitored for viability and expression of anygenes contained in the vector.

The expression vector may also contain one or more selectable markergenes that, upon expression, confer one or more phenotypic traits usefulfor selecting or otherwise identifying host cells that carry theexpression vector. Non-limiting examples of suitable selectable markersfor eukaryotic cells include dihydrofolate reductase and neomycinresistance. Non-limiting examples of suitable selectable markers forprokaryotic cells include tetracycline, ampicillin, chloramphenicol,carbenicillin, and kanamycin resistance.

For production of isoprenoid at an industrial scale, it may beimpractical or too costly to use a selectable marker that requires theaddition of an antibiotic to the fermentation media. Accordingly, someembodiments of the present invention employ host cells that do notrequire the use of an antibiotic resistance conferring selectable markerto ensure plasmid (expression vector) maintenance. In these embodimentsof the present invention, the expression vector contains a plasmidmaintenance system such as the 60-kb IncP (RK2) plasmid, optionallytogether with the RK2 plasmid replication and/or segregation system, toeffect plasmid retention in the absence of antibiotic selection (see,for example, Sia et al. (1995) J. Bacteriol. 177:2789-97; Pansegrau etal. (1994) J. Mol. Biol. 239:623-63). A suitable plasmid maintenancesystem for this purpose is encoded by the parDE operon of RK2, whichcodes for a stable toxin and an unstable antitoxin. The antitoxin caninhibit the lethal action of the toxin by direct protein-proteininteraction. Cells that lose the expression vector that harbors theparDE operon are quickly deprived of the unstable antitoxin, resultingin the stable toxin then causing cell death. The RK2 plasmid replicationsystem is encoded by the trfA gene, which codes for a DNA replicationprotein. The RK2 plasmid segregation system is encoded by the parCBAoperon, which codes for proteins that function to resolve plasmidmultimers that may arise from DNA replication.

The subject vectors can be introduced into a host cell stably ortransiently by variety of established techniques. For example, onemethod involves a calcium chloride treatment wherein the expressionvector is introduced via a calcium precipitate. Other salts, for examplecalcium phosphate, may also be used following a similar procedure. Inaddition, electroporation (that is, the application of current toincrease the permeability of cells to nucleic acids) may be used. Othertransformation methods include microinjection, DEAE dextran mediatedtransformation, and heat shock in the presence of lithium acetate. Lipidcomplexes, liposomes, and dendrimers may also be employed to transfectthe host microorganism.

Upon transformation, a variety of methods can be practiced to identifythe host cells into which the subject vectors have been introduced. Oneexemplary selection method involves subculturing individual cells toform individual colonies, followed by testing for expression of thedesired gene product. Another method entails selecting transformed hostcells based upon phenotypic traits conferred through the expression ofselectable marker genes contained within the expression vector. Those ofordinary skill can identify genetically modified host cells using theseor other methods available in the art.

The introduction of various pathway sequences of the invention into ahost cell can be confirmed by methods such as PCR, Southern blot orNorthern blot hybridization. For example, nucleic acids can be preparedfrom the resultant host cells, and the specific sequences of interestcan be amplified by PCR using primers specific for the sequences ofinterest. The amplified product is subjected to agarose gelelectrophoresis, polyacrylamide gel electrophoresis or capillaryelectrophoresis, followed by staining with ethidium bromide, SYBR Greensolution or the like, or detection of DNA with a UV detection.Alternatively, nucleic acid probes specific for the sequences ofinterest can be employed in a hybridization reaction. The expression ofa specific gene sequence can be ascertained by detecting thecorresponding mRNA via reveres-transcription coupled PCR, Northern blothybridization, or by immunoassays using antibodies reactive with theencoded gene product. Exemplary immunoassays include but are not limitedto ELISA, radioimmunoassays, and sandwich immunoassays.

The enzymatic activity of a given pathway enzyme can be assayed by avariety of methods known in the art. In general, the enzymatic activitycan be ascertained by the formation of the product or conversion of asubstrate of an enzymatic reaction that is under investigation. Thereaction can take place in vitro or in vivo. For example, the relativeactivity of HMG-CoA reductase and HMG-CoA synthase in a cell can bemeasured by the steady state level of HMG-CoA in a cell. HMG-CoA can beextracted by Tricholoroacetic Acid (TCA), followed by analyzing theextracted material via Liquid Chromatography/Mass Spectrometry. Theactivity of mevalonate kinase can be demonstrated by the formation ofmevalonate 5-phosphate. The relative activity of mevalonate kinase andHMG-CoA reductase can be measured by the steady state level ofmevalonate, which can be determined by Gas Chromatography/Massspectrometry. See e.g., WO05033287, which is incorporated herein byreference.

The yield of an isoprenoid via one or more metabolic pathways disclosedherein can be augmented by inhibiting reactions that divertintermediates from productive steps towards formation of the isoprenoidproduct. Inhibition of the unproductive reactions can be achieved byreducing the expression and/or activity of enzymes involved in one ormore unproductive reactions. Such reactions include side reactions ofthe TCA cycle that lead to fatty acid biosynthesis, alaninebiosynthesis, the aspartate superpathway, gluconeogenesis, hemebiosynthesis, and/or glutamate biosynthesis, at a level that affects theoverall yield of an isoprenoid production. Additionally, the conversionof acetyl-CoA to acetate via the action of phosphotransacetylase isanother example of unproductive side reaction. Therefore, where desired,“knocking out” or “knocking down” the pta gene that encodesphosphotransacetylase may also be carried in order to increase the yieldof isoprenoid production. Depending on the specific isoprenoid ofinterest, one skilled in the art may choose to target additionalunproductive steps. For example, where carotenoid is the isoprenoid ofchoice, one may opt to “knock out” or “knock down” one or more genesselected from the group consisting of gdhA, aceE, fdhF, yjiD, hnr oryjfP, ackA, appY, aspC, clp, clpP, clpXP, crcB, csdA, cyaA, evgS, fdhA,fdhD, feoB, fumA, glnE, glxR, gntK, hycI, lipB, lysU, modA, moeA, nadA,nuoC, nuoK, pflB, pitA, pst, pstC, pta, p-yjiD, sohA, stpA, yagR, yaiD,ybaS, ycfZ, ydeN, yebB, yedN, yfcC, ygiP, yibD, yjfP, yjhH, or yliEgenes, or any other genes alone or in combination, the inhibition ofwhich would result in a higher yield of carotenoid as described in U.S.Patent Application 20060121558, which is incorporated herein byreference.

A variety of methods are available for knocking out or knocking down agene of interest. For example, a reduced gene expression may beaccomplished by deletion, mutation, and/or gene rearrangement. It canalso be carried out with the use of antisense RNA, siRNA, miRNA,ribozymes, triple stranded DNA, and transcription and/or translationinhibitors. In addition, transposons can be employed to disrupt geneexpression, for example, by inserting it between the promoter and thecoding region, or between two adjacent genes to inactivate one or bothgenes.

High Yields of Isoprenoid Compounds

The present invention provides compositions and methods for a robustproduction of isoprenoids by the use of isopentenyl pyrophosphatepathway enzymes that are under the control of at least one heterologousregulator or fermentation conditions, either alone or in combination.

In one aspect, a method of producing an isoprenoid involves the steps of(a) obtaining a plurality of host cells that comprise an enzymaticpathway for making isopentenyl pyrophosphate wherein the all of thepathway enzymes are under control of at least one heterologoustranscriptional regulator; and (b) culturing the host cells in a mediumunder conditions that are suboptimal as compared to conditions thatwould provide for a maximum specific growth rate for the host cells. Insome embodiments, the pathway is the mevalonate pathway. In otherembodiments, the pathway is the DXP pathway. In other embodiments, theat least one heterologous transcriptional regulatory sequence isinducible. In other embodiments, the pathway enzymes are under controlof a single transcriptional regulator. In other embodiments, the pathwayenzymes are under control of multiple heterologous transcriptionalregulators.

In some embodiments, the pathway comprises a nucleic acid sequenceencoding a mevalonate pathway enzyme from a prokaryote having anendogenous mevalonate pathway. Non-limiting examples of suitableprokaryotes include those from the genera: Actinoplanes; Archaeoglobus;Bdellovibrio; Borrelia; Chloroflexus; Enterococcus; Lactobacillus;Listeria; Oceanobacillus; Paracoccus; Pseudomonas; Staphylococcus;Streptococcus; Streptomyces; Thermoplasma; and Vibrio. Non-limitingexamples of specific strains include: Archaeoglobus fulgidus;Bdellovibrio bacteriovorus; Borrelia burgdorferi; Chloroflexusaurantiacus; Enterococcus faecalis; Enterococcus faecium; Lactobacillusjohnsonii; Lactobacillus plantarum; Lactococcus lactis; Listeriainnocua; Listeria monocytogenes; Oceanobacillus iheyensis; Paracoccuszeaxanthinifaciens; Pseudomonas mevalonii; Staphylococcus aureus;Staphylococcus epidermidis; Staphylococcus haemolyticus; Streptococcusagalactiae; Streptomyces griseolosporeus; Streptococcus mutans;Streptococcus pneumoniae; Streptococcus pyogenes; Thermoplasmaacidophilum; Thermoplasma volcanium; Vibrio cholerae; Vibrioparahaemolyticus; and Vibrio vulnificus;

In another embodiment, the nucleic acid sequence encoding a mevalonatepathway enzyme is selected from acetyl-CoA thiolase, HMG-CoA synthase,HMG-CoA reductase, and mevalonate kinase. In another embodiment, thenucleic acid sequence encoding a mevalonate pathway enzyme is selectedfrom acetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, andmevalonate kinase and is from a prokaryote belonging to the genusEnterococcus or the genus Pseudomonas or the genus Staphylococcus. Inanother embodiment, the nucleic acid sequence encoding a mevalonatepathway enzyme is selected from acetyl-CoA thiolase, HMG-CoA synthase,HMG-CoA reductase, and mevalonate kinase and is from Enterococcusfaecalis or from Staphyloccoccus aureus.

In another embodiment, nucleic acid sequence encoding a mevalonatepathway enzyme is a Class II HMG-CoA reductase. HMG-CoA reductases aregenerally classified into two classes, which are distinguishable basedon sequence homology and/or enzymatic properties (see, for example,Hedl, et al., J. Bacteriology, 1927-1932, 2004, and Bochar, et al.,Molec. Genet. Metab., 66, 122-127, 1999).

Class II HMG-CoA reductases can be characterized, in part, by their lowsensitivity to statins, including but not limited to Atorvastatin,Cerivastatin, Fluvastatin, Lovastatin, Pravastatin, Simvastatin, in someembodiments, the Class II HMG-CoA reductase exhibits a statin inhibitionconstant greater than about 1 micromolar, 10 micromolar, or 100micromolar. In other embodiments, the Class II HMG-CoA reductase has aninhibition constant for Lovastatin greater than that of the Class IHMG-CoA by a factor of at least about 10, 100, 1000, or 10,000. In otherembodiments, the Class II HMG-CoA reductase has an inhibition constantfor Lovastatin greater than that of the Class I HMG-CoA isolated from aHomo sapien by a factor of at least about 1, 100, 1000, or 10,000. Inother embodiments, the Class II HMG-CoA reductase is from a prokaryote.In other embodiments, the Class II HMG-CoA reductase is from archaebacteria.

A prototypical Class II HMG-CoA reductase is derived from Pseudomonasmevalonii. Also encompassed in the invention are variant Class IIHMG-CoA reductases exhibiting at least about 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 80%, 90%, or 95% identity as compared to the aminoacid sequence of P. mevalonii HMG-CoA reductase. Further encompassed inthe invention are variants having less than about 40%, 35%, 30%, 25%,20%, or less, identity with an H. Sapiens HMG-CoA reductase. Theidentities of amino acid sequences can be determined by the methodsdescribed in Bochar, et al., Molec. Genet. Metab., 66, 122-127, 1999.

Non-limiting exemplary Class II HMG-CoA reductases include those derivedfrom HMG-CoA reductases from: Archaeoglobus fulgidus (NC_(—)000917);Bdellovibrio bacteriovorus (BX842650); Borrelia burgdorferi (AE001169);Chloroflexus aurantiacus (AJ299212); Enterococcus faecalis (AA081155);Enterococcus faecium (AF290094); Lactobacillus johnsonii (AE017204);Lactobacillus plantarum; Lactococcus lactis (AE006387); Listeria innocua(CAC96053); Listeria monocytogenes (AE017324); Oceanobacillus iheyensis(NC_(—)000917); Paracoccus zeaxanthinifaciens (AJ431696); Pseudomonasmevalonii (M24015); Staphylococcus aureus (AF290086); Staphylococcusepidermidis (AF290090); Staphylococcus haemolyticus (AF290088);Streptococcus agalactiae (CAD47046); Streptomyces griseolosporeus(AB037907); Streptococcus mutans (AAN58647); Streptococcus pneumoniae(AF290098); Streptococcus pyogenes (AF290096); Thermoplasma acidophilum(CAC11548); Thermoplasma volcanium (AL935253); Vibrio cholerae(AAF96622); Vibrio parahaemolyticus (BAC62311); and Vibrio vulnificus(AA007090).

The fermentation methods described herein relate to modulating thespecific growth rate of the host cells. Often represented by theparameter μ, the specific growth rate represents the rate of growth ofcells per unit of biomass per unit time. The specific growth rate hasthe units of reciprocal time (1/t). The maximum specific growth rate forcells in a culture medium relates to the effect of substrateconcentration on growth rate. Generally, cells will grow slowly at a lowlevel of the substrate, and as the level of the substrate in the mediumincreases, so does the rate of cell growth. However, the rate of cellgrowth does not continue to rise indefinitely, and at high levels ofsubstrate, a given increase in the amount of substrate will produce asmaller and smaller increase in the rate of cell growth. Therefore, thegrowth rate ultimately reaches a limit, which is often referred to asthe maximum specific growth rate. A theoretical treatment of therelationship between growth rate in culture is well known to thoseskilled in the art, and is referred to as the Monod equation. See, forexample, Pirt, Principles of Microbe and Cell Cultivation, Wiley, NY,1975, pages 4-10. In this theoretical treatment, the maximum specificrate is an asymptotic limit that is never reached until an infinitelevel of substrate is reached. In practice, however, the maximumspecific growth rate can be considered as being obtained when theconditions under investigation (e.g., a substrate level or temperature)support the fastest initial growth rate. For instance, in a fed-batchreactor, the initial condition where the nutrients are supplied inexcess is treated as the conditions for the maximum growth rate. See,for example, Lee et al. (1996) Trends Biotechnol. 14: 98-105 and Korz etal. (1995) J Biotechnology 39:59-65. The conditions where a substrate isadded to support the maximum specific growth rate is also sometimesreferred to as unlimited growth. In addition, while the Monod equationdescribes the theoretical rate properties for a substrate thatasymptotically approaches the maximum specific rate, for manysubstrates, rather than approaching the value as more substrate isadded, a decrease in rate is seen at higher levels of substrate after amaximum rate is achieved, i.e., the maximum specific growth rate isachieved followed by a decrease in growth rate.

The maximum specific growth rate can also be applied with respect totemperature as well as to substrates. Generally, an organism will growslowly at low temperatures, and will grow at a faster rate as thetemperature increases up to a certain point, after which, the growthrate will decline. There will be a temperature at which the growth ratewill be at a maximum level, this is the temperature at which the maximumgrowth rate is achieved. We have found that the production ofisoprenoids can be increased by lowering the temperature below thetemperature that supports the maximum specific growth rate.

The maximum specific growth rate can also be applied with respect toother additives to the fermentation than substrates. For instance, withrespect to nutrients, vitamins, and minerals, there can be a low rate atlow amounts of these components, the rate will get higher as theconcentration of the component is increased, then, in some cases, ateven higher concentrations of the components, the rate will decrease.The maximum specific growth rate is obtained where the concentration ofthe component supports the highest rate.

The maximum specific growth rate for a cell in a medium is oftendetermined at the initial stages of the fermentation before inhibitionby end product or intermediates, cell crowding, or other factorscontribute to slowing down the rate of growth. For example the maximumgrowth rate is often determined during the exponential phase of growthrather than at the lag phase, deceleration phase, or the stationaryphase. The concept of maximum specific growth rate can also be appliedat later stages of the fermentation by taking into account theappropriate variables.

Accordingly, in some embodiments, host cells are cultured underconditions such that growth is less than about 90% of the maximumspecific growth rate. In other embodiments, the host cells are culturedunder conditions such that growth is less than about 80%, 75%, 60%, 50%,40%, 30%, 25%, 20%, 10%, 5%, or 1%, or less, of the maximum specificgrowth rate.

In other embodiments, the host cells are cultured at a mediumtemperature is at least about 2° C., 4° C., 5° C., 6° C., 8° C., 10° C.,15° C., or 20° C. below the temperature that would provide for themaximum specific growth rate. By lowering the temperature growth isreduced, which in turn, reduces the formation of toxic byproducts in themedium and the generation of metabolic heat. Lowering culturetemperature also reduces cellular oxygen demand which enables highercell-densities to be obtained.

The temperature at which at which the maximum specific growth rate of ahost cell can be achieved will depend on the type of host cell selected.This can be ascertained by growing the host cells under varioustemperatures over a defined period of time to derive the relevant growthcurves. The temperature that supports the maximum specific growth ratecan be determined by comparing the slopes of growth in the respectivecurves. In the case of E. Coli, the temperature for maximum specificgrowth rate is about 37° C. Accordingly, if E. Coli is the host cell tobe used for fermentative production of isoprenoid, the fermentationtemperature is below 37° C. If S. cerevisiae is employed, thetemperature for maximum specific growth rate is about 30° C.Accordingly, if S. cerevisiae is the host cell to be used forfermentative production of isoprenoid, the fermentation temperature isbelow 30° C. Typically a desired temperature is about 2° C., 4° C., 5°C., 6, 8, 10° C., 15° C., and 20° C. below the temperature at which themaximum specific growth rate of the host cell can be achieved.

In other embodiments, the host cells are cultured in a fermentationmedium comprises a carbon source present in an amount that is lower thanthat which would provide for a maximum specific growth rate. In certainembodiments, the host cells are cultured in a medium where the carbonsource is maintained at a level to provide for less than about 90%, 80%,75%, 60%, 50%, 40%, 30%, 25%, 20%, 10%, 5%,1%, or less, of the maximumspecific growth rate. Any carbon-containing sources that are digestibleby the microorganism can be used. Non-limiting examples includecarbohydrates such as monosaccharides, oligosaccharides andpolysaccharides, organic acids such as acetic acid, propionic acid; andalcohols such as ethanol and propanol, and polyols such as glycerol.

In some embodiments, the carbon sources comprise primarilymonosaccharides or oligosaccharides. In other embodiments, the carbonsource consists essentially of monosaccharides and disaccharides. Instill other embodiments, the carbon source is essentially free ofcellulose.

Monosaccharides are the simple sugars that serve as building blocks forcarbohydrates. They are classified based on their backbone of carbon (C)atoms: trioses have three carbon atoms, tetroses four, pentoses five,hexoses six, and heptoses seven. The carbon atoms are bonded to hydrogenatoms (—H), hydroxyl groups (—OH), and carbonyl groups (—C═O), whosecombinations, order, and configurations allow a large number ofstereoisomers to exist. Pentoses include xylose, found in woodymaterials; arabinose, found in gums from conifers; ribose, a componentof RNA and several vitamins, and deoxyribose, a component of DNA.Exemplary hexoses include glucose, galactose, and fructose.Monosaccharides combine with each other and other groups to form avariety of disaccharides, and oligosaccharides. An oligosaccharide is asaccharide polymer containing a small number (typically three to ten) ofsimple sugars. They are generally found either O- or N-linked tocompatible amino acid side chains in proteins or lipid moieties. Apreferred oligosaccharide for use in the present fermentation reactionis disaccharide, including for example, sucrose, or trisaccharide suchas raffinose.

Where it is desired to have cellulose, glycan, starch, or otherpolysaccharides as the ultimate carbon source, these polysaccharides canbe first converted into monosaccharides and oligosaccharides by chemicalmeans or by enzymatic methods. For instance, cellulose can be convertedinto glucose by the enzyme cellulase. Accordingly, if polysaccharidessuch as cellulose found in the biomass (including e.g., canola, alfalfa,rice, rye, sorghum, sunflower, wheat, soybean, tobacco, potato, peanut,cotton, sweet potato, cassava, coffee, coconut, citrus trees, cocoa,tea, fruits such as, banana, fig, pineapple, guava, mango, oats, barley,vegetables, ornamentals, or conifers) is used as the ultimate carbonsource, it can be digested by cellulase to generate simpler sugars foruse in conjunction with the fermentation procedure of the presentinvention. In certain embodiments, after the breakdown of thepolysaccharide, the monosaccharide and/or oligosaccharide constitute atleast about 50% by weight of the carbon source as determined at thebeginning of the fermentation. In other embodiments, the monosaccharideand/or oligosaccharide constitute at least about 80% or even 90% byweight of the carbon source as determined at the beginning of thefermentation, such that the fermentation medium is essentially free ofcellulose.

In other embodiments, the host cells are cultured in a fermentationmedium comprises a nitrogen source present in an amount that is lowerthan that which would provide for a maximum specific growth rate. Whilenot being bound by any particular theory, it is known that changing thelevels of components such as nitrogen that are available to a cell canchange the relative flux through the various chemical pathways withinthe cell. We have found that by restricting the level of nitrogenavailable to the microorganism, the amount of isoprenoid such asamorphadiene produced by the microorganism is increased. Exemplarylevels of nitrogen of the present invention include in an amount thatwould support about 90%, 80%, 75%, 60%, 50%, 40%, 30%, 25%, 20%, 10%,5%, 1%, or less, of the maximum specific growth rate.

The restriction of nitrogen can be implemented in stages. In someembodiments, nitrogen in the form of ammonia is provided in thebeginning of the fermentation to support initial growth, but subsequentadditions to the fermentation are free of nitrogen, or are free ofnitrogen save for that level of ammonia needed to maintain the pH of thefermentation at 7 with an ammonia solution. For the bulk of thefermentation, the level of nitrogen is maintained at a level which isless than the amount which would support the maximum specific growthrate. The amounts can be for example amounts which would support atleast about 90%, 80%, 75%, 60%, 50%, 40%, 30%, 25%, 20%, 10%, 5%, or 1%,or less, of the maximum specific growth rate. A fermentation of thepresent invention could have an initial nitrogen level above 10 mM asmeasured in the fermentation medium, to support an initial growth, and asubsequent a nitrogen level below 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, or4 mM in the fermentation medium.

Sources of assimilable nitrogen that can be used in a suitablefermentation reaction mixture include, but are not limited to, simplenitrogen sources, organic nitrogen sources, and complex nitrogensources. Such nitrogen sources include anhydrous ammonia, ammonium saltsof inorganic or organic acids such as ammonium chloride, ammoniumsulfate, ammonium acetate, ammonium phosphate, other nitrogen-containingcompounds and substances of animal, vegetable, and/or microbial origin.Amino acids can also be used as the nitrogen source, including leucine,isoleucine or valine, or a mixture thereof.

Any known method for providing a substrate to a fermentation reactionmay be used to maintain the substrate level below the level which wouldprovide for the maximum specific growth rate. Illustrative examplesinclude the batch method where all the substrate for the fermentation isadded in the beginning of the fermentation reaction; the continuous feedmethod; and the variable feed rate method, where, for instance, anincreasing amount of substrate is provided as the fermentation proceedsin order to support the increased concentration of cells in the medium.Combinations of these three methods are often employed. For instance, itis common to have a certain amount of substrate present initially in thefermentation, to allow the microorganisms to deplete this initial amountof substrate, then to subsequently add substrate either continuously orto add it variably after the initial amount of substrate is utilized. Itis an aspect of this invention to provide an initial amount of substrateto the cells in the fermentation medium, which may be present at arelatively high level, to allow the host cells to substantially use upthe initial substrate, then to subsequently provide substrate to thehost cells at a level that is suboptimal as compared to the amount thatwould support the maximum growth rate by either a continuous or variablefeed rate. It will be appreciated by those of skill in the art thatsince the cells may be growing at an exponential rate, it can beadvantageous to vary the feed rate at an exponential rate in order tokeep the amount of substrate relative to the level of host cellsconstant. Thus in certain embodiments, substrates are added in anexponentially increasing manner, but yet at a level which is lower thanthe level which would provide the maximum specific growth rate.

In some embodiments, the fermentation reaction is given a reduced feedof carbon source relative to the carbon feed which would provide for themaximum specific growth rate. While not being bound by a particulartheory, it is known that changing the levels of nutrients available to acell will change the relative flux through the various chemical pathwayswithin the cell. For example, some enzymes are inducible, and will onlybe active when certain nutrients are present. We have observed thatlowering the carbon source feed rate to a microorganism can improve theamount of isoprenoid produced in the fermentation. In practice, thecarbon source can be supplied initially in an amount sufficient tosupport an initial growth of the host cells until such initial carbonsource is substantially depleted, after which the carbons source isadded at an exponential rate, but at a rate which is below that whichwould support the maximum specific growth of the host cells. Forexample, in a method of the present invention, the carbon source isadded in an amount that would support about 90%, 80%, 75%, 60%, 50%,40%, 30%, 25%, 20%, 15%, 10%, or less, of the maximum specific growthrate.

In other embodiments, the fermentation reaction is given an initialbolus of carbon source sufficient to grow at or near the maximumspecific growth rate (unlimited growth) followed by a reduced feed rateat a level below that required to support the maximum specific growthrate, for the remainder of the fermentation. In some cases, the point atwhich the reduced carbon source feed rate is implemented is the point atwhich a predetermined feed rate is achieved. In certain embodiments, themicroorganism is provided with enough carbon source to growexponentially to a feed rate of about 15 g/L/hr, after which the feedrate is reduced to 5.7 g/L/hr and held constant at that rate for theremainder of the fermentation.

In certain embodiments, one or more of the heterologous mevalonate orDXP pathway enzymes is inducible and induced after the carbon sourcefeed rate has been reduced to a level below that required for maximumspecific growth. For example, where the engineered microorganism has aninducible promoter, the fermentation is first run by adding carbonsource to achieve a exponential growth, but at a level which is belowthat to support maximum specific growth, then the carbon source feedrate is reduced to an even lower level for the remainder of thefermentation, and the inducer added after the carbon source feed rate isreduced. In some embodiments, the microorganisms are induced withisopropylthio-beta-D-galactoside (IPTG) after the reduced carbon sourcefeed is initiated.

In other embodiments, the fermentation reaction is performed in a mannerthat avoids the build up of toxic substances that decrease cell growthrates. For example, it is known that when too much glucose is added tothe medium, toxic products such as acetate can build up in the organism.See, for example, Kortz et al. (1995) J. Biotechnol. 39: 59-65. Thus byproviding a high level of carbon source, at or approaching the amountwhich would support a maximum growth rate (unlimited growth), theinitial growth of the cells may be higher, but the growth becomesarrested due to the accumulation of toxic substances. The level at whichthe carbon source is added below the level where the toxic products donot accumulate is referred to as the critical level or the inhibitorythreshold. Thus in certain embodiments, the fermentation reaction isperformed such that the carbon source is kept below the critical levelfor the build up of toxic substances. Those skilled in the art willappreciate that the critical concentration of substrates will vary withthe strain and the medium which is used.

An effective fermentation reaction mixture can contain other compoundssuch as inorganic salts, vitamins, trace metals, or growth promoters.Such other compounds can also be present in carbon, nitrogen or mineralsources in the effective reaction mixture or can be added specificallyto the reaction mixture. One embodiment of the invention involvesproviding these compounds at levels that are suboptimal as compared tothat would support the maximum growth rate of the host cells in order toincrease isoprenoid production.

The fermentation reaction mixture can also contain a suitable phosphatesource. Such phosphate sources include both inorganic and organicphosphate sources. Non-limiting examples of phosphate sources include,but are not limited to, phosphate salts such as mono or dibasic sodiumand potassium phosphates, ammonium phosphate, polyphosphate, andmixtures thereof. A suitable fermentation reaction mixture can alsoinclude a source of magnesium. In some embodiments, the magnesium is inthe form of a physiologically acceptable salt, such as magnesium sulfateheptahydrate, although other magnesium sources in concentrations thatcontribute similar amounts of magnesium can be used. Further, in someinstances it may be desirable to allow the fermentation reaction mixtureto become depleted of a magnesium source during fermentation. In someembodiments, the phosphorous source is provided in an amount that issuboptimal as compared to that would support a maximum specific growthrate.

The fermentation reaction mixture can also include a biologicallyacceptable chelating agent, such as the dihydrate of trisodium citrateand ethylenediaminetetraacetic acid. The fermentation reaction mixturecan also initially include a biologically acceptable acid or base tomaintain the desired pH of the fermentation reaction mixture.Biologically acceptable acids include, but are not limited to,hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid andmixtures thereof. Biologically acceptable bases include, but are notlimited to, ammonium hydroxide, sodium hydroxide, potassium hydroxideand mixtures thereof.

The fermentation reaction mixture can also include a biologicallyacceptable calcium source, including, but not limited to, calciumchloride. The fermentation reaction mixture can also include sodiumchloride. The fermentation reaction mixture can also include tracemetals. Such trace metals can be added to the fermentation reactionmixture as a stock solution that, for convenience, can be preparedseparately from the rest of the fermentation reaction mixture. Asuitable trace metals solution can include, but is not limited to sodiumselenate; ferrous sulfate; heptahydrate; cupric sulfate, pentahydrate;zinc sulfate, heptahydrate; sodium molybdate, dihydrate; cobaltouschloride; selenium or chromium solution; hexahydrate; and manganoussulfate monohydrate. Hydrochloric acid may be added to the stocksolution to keep the trace metal salts in solution.

If a pathway intermediate or a compound that can be converted to apathway intermediate is added to the fermentation medium, theintermediate or compound is typically present in an excess amount.

Fermentation can be conducted under anaerobic (deficient in oxygen) oraerobic (oxygenated) conditions. Under aerobic conditions,microorganisms can break down sugars to end products such as CO₂ andH₂O. Under anaerobic conditions, the host cells utilize an alternativepathway to produce CO₂ and ethanol. Fermentation can also be used torefer to the bulk growth of microorganisms on a growth medium where nodistinction is made between aerobic and anaerobic metabolism. Ingeneral, aerobic fermentation is carried out for production ofisoprenoids.

The fermentations of the present invention can be carried out in abatch, a fed-batch, or a continuous process. A batch process istypically a closed process where all of the raw materials are added atthe beginning of the fermentation. A fed-batch process is typically aclosed process where the carbon source and/or other substrates are addedin increments throughout the process. A fed-batch process allows forgreater control of the medium and the growth of the microorganisms. Acontinuous process can be considered an open system where medium iscontinuously added and product is simultaneously removed. Processes inbetween these types can also be used. For instance, in one embodiment,the fermentation is begun as a fed-batch process, and an organic layer,such as dodecane is placed in contact with the fermentation medium whilethe fermentation process continues. Isoprenoids, which typically have ahigher solubility in the organic medium than in the aqueous fermentationmedium are extracted out of the fermentation medium into the organiclayer. Where the isoprenoids are produced in excess of the saturationpoint and form a layer separable from the medium, then simple separationby way of draining or sucking the distinct phase layer can be carriedout. This process has characteristics of both a fed-batch process and acontinuous process, because of the removal of product from the mediumand the fermentation progresses. The fed-batch and continuous processesallow for the control of the addition of fermentation components duringthe fermentation process. A fed-batch, continuous, or combination ofthese processes is usually preferred in carrying out the invention. Theprocesses allow for greater control of the rate of addition of feed andother fermentation components as a function of time. The removal ofproduct during fermentation can be beneficial, especially where theaccumulated product leads to inhibition of the production pathways.

The amount of microorganism per liter of fermentation, or the density ofmicroorganism, can be measured by measuring the weight of microorganismisolated from a given volume of the fermentation medium. A commonmeasure is the dry weight of cells per liter of fermentation medium.Another method which can be used to monitor the fermentation while it isprogressing is by a measurement of the optical density of the medium. Acommon method is to measure the optical density at a wavelength of 600nm, referred to the OD₆₀₀, or the OD. The OD can be correlated to a thedensity of a specific type of organism within a specific medium, but thespecific relationship between OD and amount of microorganism per volumewill not generally be applicable across all types of organisms in alltypes of media. A calibration curve can be created by measuring the ODand the dry cell weight over a range of cell densities. In some cases,these correlations can be used in different fermentation of the same orsimilar microorganisms in the same or similar media.

In another aspect, the present invention provides a method comprisingthe steps of (i) performing a fermentation reaction comprising afermentation medium and a plurality of genetically modified host cellsthat produce the isoprenoid under conditions such that (a) thefermentation medium is kept at a temperature lower than that which wouldprovide for a maximum specific growth rate of said host cells; (b) thefermentation medium comprises a carbon source present in an amount thatis lower than that which would provide for a maximum specific growthrate of the host cells; and/or (c) the fermentation medium comprises anitrogen source present in an amount that is lower than that which wouldprovide for a maximum specific growth rate of the host cells; (ii)recovering the isoprenoid produced under one or more conditions setforth in (a) through (c). In one embodiment, the fermentation reactionis run under condition (a). In another embodiment, the fermentationreaction is run under conditions (a) and (b). In yet another embodiment,the fermentation reaction is run under conditions of (a), (b), and (c),or in any other combinations thereof.

Using the methods described herein, the host cells produce more thanabout 10 grams of isoprenoid per liter of fermentation reaction mixture(10 g/L). In other embodiments, more than about 15 g/L, more than about20 g/L, more than 25 g/L is produced, or more than about 30 g/L ofisoprenoid is produced.

In another embodiment, the host cells produce more than about 50milligrams of isoprenoid per gram of dry host cells (50 milligrams pergram dry cell weight) is produced. In other embodiments, more than about100 milligrams per gram dry cell weight, more than about 150 milligramsper gram dry cell weight, more than about 200 milligrams per gram drycell weight, more than about 250 milligrams per gram dry cell weight,more than about 500 milligrams per gram dry cell weight, more than about750 milligrams per gram dry cell weight, or more than about 1000milligrams per gram dry cell weight of isoprenoid is produced.

In other embodiments, the production level, whether it is in grams perliter or milligrams per gram dry cell weight is achieved in less thanabout 150 hours, preferably less than about 96 hours, or even less thanabout 72 hours.

Non-limiting examples of suitable isoprenoids include: hemiterpenes(derived from 1 isoprene unit) such as isoprene; monoterpenes (derivedfrom 2 isoprene units) such as myrcene; sesquiterpenes (derived from 3isoprene units) such as amorpha-4,11-diene; diterpenes (derived fromfour isoprene units) such as taxadiene; triterpenes (derived from 6isoprene units) squalene; tetraterpenes (derived from 8 isoprenoids)β-carotene; and polyterpenes (derived from more than 8 isoprene units)such as polyisoprene. In some embodiments, the isoprenoid is not acarotenoid. In other embodiments, the isoprenoid is a C₅-C₂₀ isoprenoid.

Although the invention has been described in conjunction with specificembodiments thereof, the foregoing description is intended to illustrateand not limit the scope of the invention. Other aspects, advantages, andmodifications within the scope of the invention will be apparent tothose skilled in the art to which the invention pertains.

EXAMPLES

The practice of the present invention can employ, unless otherwiseindicated, conventional techniques of the biosynthetic industry and thelike, which are within the skill of the art. To the extent suchtechniques are not described fully herein, one can find ample referenceto them in the scientific literature.

In the following examples, efforts have been made to ensure accuracywith respect to numbers used (for example, amounts, temperature, and soon), but variation and deviation can be accommodated, and in the event aclerical error in the numbers reported herein exists, one of ordinaryskill in the arts to which this invention pertains can deduce thecorrect amount in view of the remaining disclosure herein. Unlessindicated otherwise, temperature is reported in degrees Celsius, andpressure is at or near atmospheric pressure at sea level. All reagents,unless otherwise indicated, were obtained commercially. The followingexamples are intended for illustrative purposes only and do not limit inany way the scope of the present invention.

Example 1

This example describes methods for making expression plasmids thatencode for enzymes including enzymes of the MEV pathway fromSaccharomyces cerevisiae organized in operons.

Expression plasmid pMevT was generated by inserting the MevT operon (SEQID NO: 1) into the pBAD33 vector. The MevT operon encodes the set of MEVpathway enzymes that together transform the ubiquitous precursoracetyl-CoA to (R)-mevalonate, namely acetoacetyl-CoA thiolase, HMG-CoAsynthase, and HMG-CoA reductase. The MevT operon was generated by PCRamplifying from Escherichia coli genomic DNA the coding sequence of theatoB gene (GenBank accession number NC_(—)000913 REGION: 2324131 . . .2325315) (encodes an acetoacetyl-CoA thiolase), from Saccharomycescerevisiae genomic DNA the coding sequence of the ERG13 gene (GenBankaccession number X96617, REGION: 220.1695) (encodes a HMG-CoA synthase),and from Saccharomyces cerevisiae genomic DNA a segment of the codingregion of the HMG1 gene (GenBank accession number M22002, REGION: 1660 .. . 3165) (encodes a truncated HMG-CoA reductase (tHMGR)). The upstreamPCR primer used for the amplification of the HMG1 gene fragment includedan artificial start codon. The amplified fragments were spliced togetherusing overlap extensions (SOEing), during which process ribosome bindingsites were introduced after the atoB and the ERG13 coding sequences.After the addition of 3′ A overhangs, the MevT operon was ligated intothe TA cloning vector pCR4 (Invitrogen, Carlsbad, Calif.), and sequencedto ensure accuracy. The MevT operon was subsequently ligated into theXmaI PstI restriction enzyme site of vector pBAD33 (Guzman et al. (1995)J. Bacteriol. 177(14): 4121-4130). To place the operon under the controlof the P_(Lac) promoter, the araC-P_(BAD)NsiI-XmaI fragment of pBAD33was replaced with the NsiI-XmaI fragment of pBBR1MCS, yieldingexpression plasmid pMevT (see U.S. Pat. No. 7,192,751).

Expression plasmid pAM36-MevT66 was generated by inserting the MevT66operon into the pAM36 vector. Vector pAM36 was generated by inserting anoligonucleotide cassette containing AscI-SfiI-AsiSI-XhoI-PacI-FsIl-PmeIrestriction enzyme sites into the pACYC184 vector (GenBank accessionnumber X06403), and by removing the tet resistance gene in pACYC184. TheMevT66 operon was synthetically generated using the nucleotide sequenceSEQ ID NO: 1 as a template, which comprises the atoB gene fromEscherichia coli (GenBank accession number NC_(—)000913 REGION: 2324131. . . 2325315), the ERG13 gene from Saccharomyces cerevisiae (GenBankaccession number X96617, REGION: 220.1695), and a truncated version ofthe HMG1 gene from Saccharomyces cerevisiae (GenBank accession numberM22002, REGION: 1777 . . . 3285), all three sequences beingcodon-optimized for expression in Escherichia coli. The syntheticallygenerated MevT66 operon was flanked by a 5′ EcoRI restriction enzymesite and a 3′ Hind III restriction enzyme site, and could thus be clonedinto compatible restriction enzyme sites of a cloning vector such as astandard pUC or pACYC origin vector. From this construct, the MevT66operon was PCR amplified with flanking SfiI and AsiSI restriction enzymesites, the amplified DNA fragment was digested to completion using SfiIand AsiSI restriction enzymes, the reaction mixture was resolved by gelelectrophoresis, the approximately 4.2 kb DNA fragment was gel extractedusing a Qiagen gel purification kit (Valencia, Calif.), and the isolatedDNA fragment was ligated into the SfiI AsiSI restriction enzyme site ofthe pAM36 vector, yielding expression plasmid pAM36-MevT66.

Expression plasmid pAM25 was generated by inserting the MevT66 operoninto the pAM29 vector. Vector pAM29 was created by assembling the p15Aorigin of replication and kan resistance gene from pZS24-MCS1 (Lutz andBujard (1997) Nucl Acids Res. 25:1203-1210) with anoligonucleotide-generated lacUV5 promoter. The DNA synthesis constructcomprising the MevT66 operon (see above) was digested to completionusing EcoRI and Hind III restriction enzymes, the reaction mixture wasresolved by gel electrophoresis, the 4.2 kb DNA fragment was gelextracted, and the isolated DNA fragment was ligated into the EcoRIHindIII restriction enzyme site of pAM29, yielding expression plasmidpAM25.

Expression plasmid pMevB-Cm was generated by inserting the MevB operoninto the pBBR1MCS-1 vector. The MevB operon encodes the set of enzymesthat together convert (R)-mevalonate to IPP, namely mevalonate kinase,phosphomevalonate kinase, and mevalonate pyrophosphate carboxylase. TheMevB operon was generated by PCR amplifying from Saccharomycescerevisiae genomic DNA the coding sequences of the ERG12 gene (GenBankaccession number X55875, REGION: 580.1911) (encodes a mevalonatekinase), the ERG8 gene (GenBank accession number Z49939, REGION: 3363 .. . 4718) (encodes a phosphomevalonate kinase), and the MVD1 gene(GenBank accession number X97557, REGION: 544.1734) (encodes amevalonate pyrophosphate carboxylase), and by splicing the PCR fragmentstogether using overlap extensions (SOEing). By choosing appropriateprimer sequences, the stop codons of ERG12 and ERG8 were changed fromTAA to TAG during amplification to introduce ribosome binding sites.After the addition of 3′ A overhangs, the MevB operon was ligated intothe TA cloning vector pCR4 (Invitrogen, Carlsbad, Calif.). The MevBoperon was excised by digesting the cloning construct to completionusing PstI restriction enzyme, resolving the reaction mixture by gelelectrophoresis, gel extracting the 4.2 kb DNA fragment, and ligatingthe isolated DNA fragment into the PstI restriction enzyme site ofvector pBBR1MCS-1 (Kovach et al., Gene 166(1): 175-176 (1995)), yieldingexpression plasmid pMevB-Cm.

Expression plasmid pMBI was generated by inserting the MBI operon intothe pBBR1MCS-3 vector. The MBI operon encodes the same enzymes as theMevB operon, as well as an isopentenyl pyrophosphatase isomerase thatcatalyzes the conversion of IPP to DMAPP. The MBI operon was generatedby PCR amplifying from Escherichia coli genomic DNA the coding sequenceof the idi gene (GenBank accession number AF 119715) using primers thatcontained an XmaI restriction enzyme site at their 5′ ends, digestingthe amplified DNA fragment to completion using XmaI restriction enzyme,resolving the reaction mixture by gel electrophoresis, gel extractingthe 0.5 kb fragment, and ligating the isolated DNA fragment into theXmaI restriction enzyme site of expression plasmid pMevB-Cm, therebyplacing idi at the 3′ end of the MevB operon. The MBI operon wassubcloned into the SalI and SacI restriction enzyme sites of vectorpBBR1MCS-3 (Kovach et al., Gene 166(1): 175-176 (1995)), yieldingexpression plasmid pMBI (see U.S. Pat. No. 7,192,751).

Expression plasmid pMBIS was generated by inserting the ispA gene intopMBI. The ispA gene encodes a farnesyl pyrophosphate synthase thatcatalyzes the condensation of two molecules of IPP with one molecule ofDMAPP to make farnesyl pyrophosphate (FPP). The coding sequence of theispA gene (GenBank accession number D00694, REGION: 484 . . . 1383) wasPCR amplified from Escherichia coli genomic DNA using a forward primerwith a SacII restriction enzyme site and a reverse primer with a SacIrestriction enzyme site. The amplified PCR product was digested tocompletion with SacII and SacI restriction enzymes, the reaction mixturewas resolved by gel electrophoresis, and the 0.9 kb DNA fragment was gelextracted. The isolated DNA fragment was ligated into the SacII SacIrestriction enzyme site of pMBI, thereby placing the ispA gene 3′ of idiand the MevB operon, and yielding expression plasmid pMBIS (see U.S.Pat. No. 7,192,751).

Expression plasmid pMBIS-gpps was derived from expression plasmid pMBISby replacing the ispA coding sequence with a nucleotide sequenceencoding a geranyl diphosphate synthase (“gpps”). A DNA fragmentcomprising a nucleotide sequence encoding the geranyl diphosphatesynthase was generated synthetically using the coding sequence of thegpps gene of Arabidopsis thaliana (GenBank accession number Y17376,REGION: 52.1320), codon-optimized for expression in Escherichia coli, asa template. The nucleotide sequence was flanked by a leader SacIIrestriction enzyme site and a terminal SacI restriction enzyme site, andcan be cloned into compatible restriction enzyme sites of a cloningvector such as a standard pUC or pACYC origin vector. The syntheticallygenerated geranyl diphosphate synthase sequence was isolated bydigesting the DNA synthesis construct to completion using SacII and SacIrestriction enzymes, resolving the reaction mixture by gelelectrophoresis, gel extracting the approximately 1.3 kb DNA fragment,and ligating the isolated DNA fragment into the SacII SacI restrictionenzyme site of expression plasmid pMBIS, yielding expression plasmidpMBIS-gpps (see FIG. 3 for a plasmid map).

Expression plasmid pAM45 was generated by inserting the MBIS operon intopAM36-MevT66 and adding lacUV5 promoters in front of the two operons.The MBIS operon was PCR amplified from pMBIS using primers comprising a5′ XhoI restriction enzyme site and a 3′ PacI restriction enzyme site.The amplified PCR product was digested to completion using XhoI and PacIrestriction enzymes, the reaction mixture was resolved by gelelectrophoresis, the 5.4 kb DNA fragment was gel extracted, and theisolated DNA fragment was ligated into the XhoI Pad restriction enzymesite of pAM36-MevT66, yielding plasmid pAM43. A DNA fragment comprisinga nucleotide sequence encoding the lacUV5 promoter was synthesized fromoligonucleotides and sub-cloned into the AscI SfiI and AsiSI XhoIrestriction enzyme sites of pAM43, yielding expression plasmid pAM45.

Example 2

This example describes methods for making expression vectors encodingenzymes including enzymes of the MEV pathway from Staphylococcus aureusorganized in operons.

Expression plasmid pAM41 was derived from expression plasmid pAM25 byreplacing the coding sequence of the HMG gene, which encodes theSaccharomyces cerevisiae HMG-CoA reductase, with the coding sequence ofthe mvaA gene, which encodes the Staphylococcus aureus HMG-CoA reductase(GenBank accession number BA000017, REGION: 2688925.2687648). The codingsequence of the mvaA gene was PCR amplified from Staphyloccoccus aureussubsp. aureus (ATCC 70069) genomic DNA using primers 4-49 mvaA SpeI (SEQID NO: 2) and 4-49 mvaAR XbaI (SEQ ID NO: 3), the amplified DNA fragmentwas digested to completion using SpeI restriction enzyme, the reactionmixture was resolved by gel electrophoresis, and the approximately 1.3kb DNA fragment was gel extracted. The HMG1 coding sequence was removedfrom pAM25 by digesting the plasmid to completion using HindIIIrestriction enzyme. The terminal overhangs of the resulting linear DNAfragment were blunted using T4 DNA polymerase. The DNA fragment was thenpartially digested using SpeI restriction enzyme, the reaction mixturewas resolved by gel electrophoresis, and the 4.8 kb DNA fragment was gelextracted. The isolated DNA fragment was ligated with the SpeI-digestedmvaA PCR product, yielding expression plasmid pAM41. The nucleotidesequence of the atoB(opt):ERG13(opt):mvaA operon contained in pAM41 isSEQ ID NO: 41.

Expression plasmid pAM52 was derived from expression plasmid pAM41 byreplacing the coding sequence of the ERG13 gene, which encodes theSaccharomyces cerevisiae HMG-CoA synthase, with the coding sequence ofthe mvaS gene, which encodes the Staphylococcus aureus HMG-CoA synthase(GenBank accession number BA000017, REGION: 2689180.2690346). ERG13 isalso known as HMGS or HMG-CoA synthase. The coding sequence of the mvaSgene was PCR amplified from Staphyloccoccus aureus subsp. aureus (ATCC70069) genomic DNA using primers HMGS 5′ Sa mvaS-S (SEQ ID NO: 4) andHMGS 3′ Sa mvaS-AS (SEQ ID NO: 5), and the amplified DNA fragment wasused as a PCR primer to replace the coding sequence of the HMG1 gene inpAM41 according to the method of Geiser et al. (BioTechniques 31:88-92(2001)), yielding expression plasmid pAM52. The nucleotide sequence ofthe atoB(opt):mvaS:mvaA operon contained in pAM52 is SEQ ID NO: 42.

Expression plasmid pAM97 was derived from expression plasmid pAM45 byreplacing the MevT66 operon with the (atoB(opt):mvaS:mvaA) operon ofexpression plasmid pAM52. Expression plasmid pAM45 was digested tocompletion using AsiSI and SfiI restriction enzymes, the reactionmixture was resolved by gel electrophoresis, and the 8.3 kb DNA fragmentlacking the MevT66 operon was gel extracted. The (atoB(opt):mvaS:mvaA)operon of pAM52 was PCR amplified using primers 19-25 atoB SfiI-S (SEQID NO: 6) and 19-25 mvaA-AsiSI-AS (SEQ ID NO: 7), the PCR product wasdigested to completion using SfiI and AsiSI restriction enzymes, thereaction mixture was resolved by gel electrophoresis, the 3.7 kb DNAfragment was gel extracted, and the isolated DNA fragment was ligatedinto the AsiSI SfiI restriction enzyme site of expression plasmid pAM45,yielding expression plasmid pAM97.

Expression plasmid pAM97-MBI was derived from expression plasmid pAM97and pAM45 by replacing the MBIS operon of pAM97 with the MBI operon ofpAM45. The MBI operon was PCR amplified from pAM45 using primers 9-70C(SEQ ID NO: 8) and 26-39B (SEQ ID NO: 9), the reaction mixture wasresolved by gel electrophoresis, the 4.5 kb DNA fragment was gelextracted, and the isolated DNA fragment was digested to completionusing SacI and XhoI restriction enzymes. Expression plasmid pAM97 wasdigested to completion using SacI and XhoI restriction enzymes, thereaction mixture was resolved by gel electrophoresis, the 7.6 kbfragment was gel extracted, and the isolated DNA fragment was ligatedwith the MBI operon PCR product, yielding expression plasmid pAM97-MBI.

Expression plasmid pAM97-MevB was derived from expression plasmid pAM97and pAM45 by replacing the MBIS operon of pAM97 with the MevB operon ofpAM45. The MevB operon was PCR amplified from pAM45 using primers 9-70C(SEQ ID NO: 8) and 26-39A (SEQ ID NO: 10), the reaction mixture wasresolved by gel electrophoresis, the 3.9 kb DNA fragment was gelextracted, and the isolated DNA fragment was digested to completionusing SacI and XhoI restriction enzymes. Expression plasmid pAM97 wasdigested to completion using SacI and XhoI restriction enzymes, thereaction mixture was resolved by gel electrophoresis, the 7.6 kbfragment was gel extracted, and the isolated DNA fragment was ligatedwith the MevB operon PCR product, yielding expression plasmidpAM97-MevB.

Expression plasmid pAM128 was generated by inserting the(atoB(opt):mvaS:mvaA) and MBIS operons of expression plasmid pAM97 intoa vector that comprises the RK2 plasmid replication, segregation, andmaintenance system, which obviates the continuous need for antibioticselection of host cell transformants. The RK2 plasmid was digested tocompletion using PstI restriction enzyme, the reaction mixture wasresolved by gel electrophoresis, the approximately 6.3 kb DNA fragmentcontaining the entire par locus was gel extracted, and the isolated DNAfragment was subcloned into the PstI restriction enzyme site of the miniRK2 replicon pRR10 (Roberts et al. (1990) J. Bacteriol. 172(11):6204-6216), yielding vector pAM132. Expression plasmid pAM97 wasdigested to completion using AscI and Sacl restriction enzymes, thereaction mixture was resolved by gel electrophoresis, the approximately9.4 kb DNA fragment was gel extracted, and the isolated DNA fragment wasligated into the MluI SacI restriction enzyme site of pAM132, yieldingexpression plasmid pAM128.

Example 3

This example describes methods for making expression vectors that encodeenzymes including enzymes of the MEV pathway from Enterococcus faecalisorganized in operons.

Plasmid pAM16 was generated by inserting the coding sequence of the mvaEgene of Enterococcus faecalis (GenBank accession number AF290092 REGION:1479 . . . 3890) (encodes an acetyl-CoA acetyltransferase/HMG-CoAreductase (HMGR)) into the pBlueScripII-KS(+) vector. The codingsequence of the mvaE gene was PCR amplified from Enterococcus faecalisgenomic DNA (ATCC 700802) using 5′ phosphorylated primers 4-40 mvaEFBamHI (SEQ ID NO: 11) and 4-40 mvaERHindIII (SEQ ID NO: 12). (Note thatprimer 4-40 mvaEF BamHI changes the start codon of the mvaE gene fromTTG to ATG in the amplified PCR product). The resulting PCR product wasligated into the SmaI restriction enzyme site of pBlueScripII-KS(+)(Stratagene, La Jolla, Calif.), yielding expression plasmid pAM16.

Plasmid pAM18 was generated by inserting the coding sequence of the mvaSgene of Enterococcus faecalis (GenBank accession number AF290092 REGION:142.1293) (encodes a HMG-CoA synthase (HMGS)) into thepBlueScripII-KS(+) vector. The coding sequence of the mvaS gene was PCRamplified from Enterococcus faecalis genomic DNA (ATCC 700802) using 5′phosphorylated primers 4-40 mvaSF BglII (SEQ ID NO: 13) and 4-39 mvaSRBamHI (SEQ ID NO: 14), and the PCR product was ligated into the SmaIrestriction enzyme site of pBlueScripII-KS(+) (Stratagene, La Jolla,Calif.), yielding expression plasmid pAM18.

Expression plasmid pAM22 was generated by inserting the coding sequenceof the mvaE gene of expression plasmid pAM16 into the pZE21-P_(L-lacO1)vector. Vector pZE21-P_(L-lacO1) is a derivative of vector pZE21-MCS-1in which the tet promoter was replaced with the P_(L-lacO1) promoter(Lutz and Bujard (1997) Nucl Acids Res. 25:1203-1210). Expressionplasmid pAM16 was digested to completion using BamHI and HindIIIrestriction enzymes, the reaction mixture was resolved by gelelectrophoresis, the approximately 2.4 kb DNA fragment containing themvaE coding sequence was gel extracted, and the isolated DNA fragmentwas inserted into the BamHI HindIII restriction enzyme site ofpZE21P_(L-lacO1), yielding expression plasmid pAM22.

Expression plasmid pAM33 was generated by inserting the coding sequenceof the mvaS gene of expression plasmid pAM18 into expression plasmidpAM22. Expression plasmid pAM18 was digested to completion using BglIIand BamHI restriction enzymes, the reaction mixture was resolved by gelelectrophoresis, the approximately 1.2 kb DNA fragment containing thecoding sequence of the mvaS gene was gel extracted, and the isolated DNAfragment was inserted into the BamHI site of expression plasmid pAM22,yielding expression plasmid pAM33.

Expression plasmid pAM34 was generated by inserting the mvaS-mvaE operonof expression plasmid pAM33 into vector pAM29. The mvaS-mvaE operon wasisolated by partially digesting pAM33 using EcoRI restriction enzyme,digesting the resulting linear DNA fragment using MluI restrictionenzyme, resolving the reaction mixture by gel electrophoresis, and gelextracting the approximately 3.6 kb DNA fragment. The vector backbone ofpAM29 was obtained by digesting to completion expression vector pAM25using MluI and EcoRI restriction enzymes, resolving the reaction mixtureby gel electrophoresis, and gel extracting the approximately 2.1 kb DNAfragment. The two isolated DNA fragments were ligated, yieldingexpression plasmid pAM34.

Example 4

This example describes methods for making expression plasmids thatencode enzymes, including enzymes of the DXP pathway from Escherichiacoli organized in operons.

Expression plasmid pAM408 was generated by inserting genes encodingenzymes of the “top” DXP pathway into the pAM29 vector. Enzymes of the“top” DXP pathway include 1-deoxy-D-xylulose-5-phosphate synthase(encoded by the dxs gene of Escherichia coli),1-deoxy-D-xylulose-5-phosphate reductoisomerase (encoded by the dxr geneof Escherichia coli), 4-diphosphocytidyl-2C-methyl-D-erythritol synthase(encoded by the ispD gene of Escherichia coli), and4-diphosphocytidyl-2C-methyl-D-erythritol synthase (encoded by the ispEgene of Escherichia coli), which together transform pyruvate andD-glyceraldehyde-3-phosphate to4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate. DNA fragmentscomprising nucleotide sequences that encode enzymes of the “top” DXPpathway were generated by PCR amplifying the coding sequences of the dxs(GenBank accession number U00096 REGION: 437539.439401), dxr (GenBankaccession number U00096 REGION: 193521.194717), ispD (GenBank accessionnumber U00096 REGION: 2869803.2870512), and ispE (GenBank accessionnumber U00096 REGION 1261249.1262100) genes from Escherichia coli strainDH1 (ATCC #33849) with added optimal Shine Dalgarno sequences and 5′ and3′ restriction enzyme sites using the PCR primers shown in SEQ ID NOS:15-18. The PCR products were resolved by gel electrophoresis, gelextracted using a Qiagen (Valencia, Calif.) gel purification kit,digested to completion using appropriate restriction enzymes Ma and KpnIfor the PCR product comprising the dxs gene; KpnI and ApaI for the PCRproduct comprising the dxr gene; ApaI and NdeI for the PCR productcomprising the ispD gene; NdeI and MluI for the PCR product comprisingthe ispE gene), and purified using a Qiagen (Valencia, Calif.) PCRpurification kit. Roughly equimolar amounts of each PCR product werethen added to a ligation reaction to assemble the individual genes intoan operon. From this ligation reaction, 1 μl of reaction mixture wasused to PCR amplify 2 separate gene cassettes, namely the dxs-dxr andthe ispD-ispE gene cassettes. The dxs-dxr gene cassette was PCRamplified using primers 67-1A-C (SEQ ID NO: 15) and 67-1D-C (SEQ ID NO:18), and the ispD-ispE gene cassette was PCR amplified using primers67-1E-C (SEQ ID NO: 19) and 67-1H-C (SEQ ID NO: 22). The two PCRproducts were resolved by gel electrophoresis, and gel extracted. ThePCR product comprising the dxs-dxr gene cassette was digested tocompletion using XhoI and ApaI restriction enzymes, and the PCR productcomprising the ispD-ispE gene cassette was digested to completion usingApaI and MluI restriction enzymes, and the two PCR products werepurified. Vector pAM29 was digested to completion using SalI and MluIrestriction enzymes, and the two digested PCR products containing the“top” DXP pathway operon were ligated into the SalI MluI restrictionenzyme site of the pAM29 vector, yielding expression plasmid pAM408 (seeFIG. 4 for a plasmid map).

Expression plasmid pAM409 was generated by inserting genes encodingenzymes of the “bottom” DXP pathway into the pAM369 vector. Enzymes ofthe “bottom” DXP pathway include 2C-methyl-D-erythritol2,4-cyclodiphosphate synthase (encoded by the ispF gene of Escherichiacoli), 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (encodedby the ispG gene of Escherichia coli), and isopentenyl/dimethylallyldiphosphate synthase (encoded by the ispH gene of Escherichia coli),which together transform4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate to IPP and DMAPP.IPP is also converted to DMAPP through the activity of isopentyldiphosphate isomerase (encoded by the idi gene of Escherichia coli).DMAPP can be further converted to FPP through the activity of farnesyldiphosphate synthase (encoded by the ispA gene of Escherichia coli). Anoperon encoding enzymes of the “bottom” DXP pathway as well as anisopentyl diphosphate isomerase and a farnesyl diphosphate synthase wasgenerated by PCR amplifying the ispF (GenBank accession number U00096REGION: 2869323 . . . 2869802), ispG (GenBank accession number U00096REGION: 2638708 . . . 2639826), ispH (GenBank accession number U00096REGION: 26277 . . . 27227), idi (GenBank accession number AF119715), andispA (GenBank accession number D00694 REGION: 484.1383) genes fromEscherichia coli strain DH1 (ATCC #33849) with added optimal ShineDalgarno sequences and 5′ and 3′ restriction enzyme sites using theappropriate PCR primers. The PCR products were resolved by gelelectrophoresis, gel extracted, digested with the appropriaterestriction enzymes (BamHI and ApaI for the PCR product comprising theispF gene; KpnI and ApaI for the PCR product comprising the ispG gene;SalI and KpnI for the PCR product comprising the ispH gene; SalI andHindIII for the PCR product comprising the idi gene; HindIII and NcoIfor the PCR product comprising the ispA gene), and purified. Roughlyequimolar amounts of each PCR product were then added to a ligationreaction to assemble the individual genes into an operon. From thisligation reaction, 1 μl of reaction mixture was used to PCR amplify 2separate gene cassettes, namely the ispF-ispG and the ispH-idi-ispA genecassettes. The ispF-ispG gene cassette was PCR amplified using primers67-2A-C (SEQ ID NO: 23) and 67-2D-C (SEQ ID NO: 26), and theispH-idi-ispA gene cassette was PCR amplified using primers 67-2E-C (SEQID NO: 27) and 67-2J-C (SEQ ID NO: 32). The two PCR products wereresolved by gel electrophoresis, and gel extracted. The PCR productcomprising the ispF-ispG gene cassette was digested to completion usingBamHI and KpnI restriction enzymes, and the PCR product comprising theispH-idi-ispA gene cassette was digested to completion using KpnI andNcoI restriction enzymes, and the two PCR products were purified. VectorpAM369 was created by assembling the p15A origin of replication frompAM29 and beta-lactamase gene for ampicillin resistance from pZE12-luc(Lutz and Bujard (1997) Nucl Acids Res. 25:1203-1210) with anoligonucleotide-generated lacUV5 promoter. Vector pAM369 was digested tocompletion using BamHI and NcoI restriction enzymes, and the 2 isolatedPCR products containing the “bottom” DXP pathway operon were ligatedinto the BamHI NcoI restriction enzyme site of the pAM369 vector,yielding expression plasmid pAM409.

Expression plasmid pAM424, a derivative of expression plasmid pAM409containing the broad-host range RK2 origin of replication, was generatedby transferring the lacUV5 promoter and the ispFGH-idi-ispA operon ofpAM409 to the pAM257 vector. Vector pAM257 was generated as follows: theRK2 par locus was PCR-amplified from RK2 plasmid DNA (Meyer et al.(1975) Science 190:1226-1228) using primers 9-156A (SEQ ID NO: 33) and9-156B (SEQ ID NO: 34), the 2.6 kb PCR product was digested tocompletion using AatII and XhoI restriction enzymes, and the DNAfragment was ligated into a plasmid containing the p15 origin ofreplication and the chloramphenicol resistance gene from vectorpZA31-luc (Lutz and Bujard (1997) Nucl Acids Res. 25:1203-1210),yielding plasmid pAM37-par; pAM37-par was digested to completion usingrestriction enzymes SacI and HindIII, the reaction mixture was resolvedby gel electrophoresis, the DNA fragment comprising the RK2 par locusand the chloramphenicol resistance gene was gel extracted, and theisolated DNA fragment was ligated into the SacI HindIII site of themini-RK2 replicon pRR10 (Roberts et al. (1990) J. Bacteriol.172:6204-6216), yielding vector pAM133; pAM133 was digested tocompletion using BglII and HindIII restriction enzymes, the reactionmixture was resolved by gel electrophoresis, the approximately 6.4 kbDNA fragment lacking the ampicillin resistance gene and oriT conjugativeorigin was gel extracted, and the isolated DNA fragment was ligated witha synthetically generated DNA fragment comprising a multiple cloningsite that contained PciI and XhoI restriction enzyme sites, yieldingvector pAM257. Expression plasmid pAM409 was digested to completionusing XhoI and PciI restriction enzymes, the reaction mixture wasresolved by gel electrophoresis, the approximately 4.4 kb DNA fragmentwas gel extracted. Vector pAM257 was digested to completion usingrestriction enzymes XhoI and PciI, and the isolated DNA fragmentcontaining the lacUV5 promoter and ispFGH-idi-ispA operon was ligatedinto the XhoI PciI restriction enzyme site of the pAM257 vector,yielding expression plasmid pAM424 (see FIG. 5 for a plasmid map).

Example 5

This example describes methods for making expression plasmids thatencode enzymes that convert FPP or GPP.

Expression plasmid pTrc99A-ADS was generated by inserting a nucleotidesequence encoding an amorpha-4,11-diene synthase (“ADS”) into vectorpTrc99A. The amorpha-4,11-diene synthase sequence was generatedsynthetically, so that upon translation the amino acid sequence would beidentical to that described by Merke et al. (2000) Ach. Biochem.Biophys. 381:173-180, so that the nucleotide sequence encoding theamorpha-4,11-diene synthase was optimized for expression in Escherichiacoli, and so that the nucleotide sequence was flanked by a 5′ NcoI and a3′ XmaI restriction enzyme site (see U.S. Pat. No. 7,192,751). Thenucleotide sequence was digested to completion using NcoI and XmaIrestriction enzymes, the reaction mixture was resolved by gelelectrophoresis, the approximately 1.6 kb DNA fragment wasgel-extracted, and the isolated DNA fragment was inserted into the NcoIXmaI restriction enzyme site of the pTrc99A vector (Amman et al. (1985)Gene 40:183-190), yielding expression plasmid pTrc99A-ADS (see FIG. 6for a plasmid map).

Expression plasmid pAM113 is a chloramphenicol-resistant derivative ofpTrc99A-ADS. It was generated by PCR amplifying the chloramphenicolresistance gene from vector pZA31-luc (Lutz and Bujard (1997) Nucl AcidsRes. 25:1203-1210) using 5′-phosphorylated primers 19-137 cml-pAM37-AS(SEQ ID NO: 35) and 19-137 cml-pAM37-S (SEQ ID NO: 36), and insertingthe 920 bp PCR product into the FspI restriction enzyme site ofexpression plasmid pTrc99A-ADS, yielding expression plasmid pAM113.

Expression plasmid pC9 was generated by inserting a genomic DNA fragmentof Bacillus subtilis 6051 comprising the coding sequence of the nudFgene and upstream genomic sequences (GenBank accession number Z99116REGION: 49364.48548) into vector pTrc99A (Amann et al. (1988) Gene69:301-315). Expression plasmid pNudF-H was generated by inserting thecoding sequence of the Bacillus subtilis 6051 nudF gene (GenBankaccession number Z99116 REGION: 49105.48548) into vector pTrc99A.Expression plasmid pyhfR was generated by inserting the coding sequenceof the Bacillus subtilis 6051 yhfR gene (GenBank accession number Z99109REGION: 97583.97002) into vector pTrc99A.

Expression plasmid pAM373 was generated by inserting a nucleotidesequence encoding the β-farnesene synthase (“FSB”) of Artemisia annua(GenBank accession number AY835398), codon-optimized for expression inEscherichia coli, into the pTrc99A vector. The nucleotide sequenceencoding the β-farnesene synthase was generated synthetically, and wasamplified by PCR from its DNA synthesis construct using the appropriateprimers. To create a leader NcoI restriction enzyme site in the PCRproduct comprising the β-farnesene synthase coding sequence, the codonencoding the second amino acid in the original polypeptide sequence (TCGcoding for serine) was replaced by a codon encoding aspartic acid (GAC)in the 5′ PCR primer (SEQ ID NO: 37). The resulting PCR product waspartially digested using NcoI restriction enzyme, and digested tocompletion using SacI restriction enzyme, the reaction mixture wasresolved by gel electrophoresis, the approximately 1.7 kb DNA fragmentcomprising the β-farnesene synthase coding sequence was gel extracted,and the isolated DNA fragment was ligated into the NcoI SacI restrictionenzyme site of the pTrc99A vector, yielding expression plasmid pAM373(see FIG. 6 for a plasmid map).

Expression plasmids pTrc99A-FSA, pTrc99A-GTS, pTrc99A-PS, pTrc99A-TSwere generated by inserting a DNA fragment comprising a nucleotidesequence encoding an α-farnesene synthase (“FSA”), a γ-terpinenesynthase (“GTS”), an α-pinene synthase (“APS”), or a terpinolenesynthase (“TS”) into the pTrc99A vector. The DNA fragment insert wasgenerated synthetically, using as a template for example the codingsequence of the α-farnesene synthase gene of Picea abies (GenBankaccession number AY473627, REGION: 24.1766), the coding sequence of theβ-farnesene synthase gene of Artemisia annua (GenBank accession numberAY835398), the coding sequence of the γ-terpinene synthase gene ofCitrus limon (GenBank accession number AF514286 REGION: 30 . . . 1832),the coding sequence of the α-pinene synthase gene of Abies grandis(GenBank accession number U87909, REGION: 6.1892) or of Pinus taeda(GenBank accession number AF543530 REGION: 1.1887), or the codingsequence of the terpinolene synthase gene of Ocimum basilicum (GenBankaccession number AY693650) or of Pseudotsuga menziesii (GenBankaccession number AY906866 REGION:10 . . . 1887) or of Abies grandis(GenBank accession number AF139206), all nucleotide sequences beingcodon-optimized for expression in Escherichia coli. The DNA fragmentsfor FSA was amplified by PCR from its DNA synthesis construct using theprimer sequences SEQ ID NO: 39 and SEQ ID NO: 40. The resulting PCRproduct was digested to completion using NcoI and SacI restrictionenzymes, the reaction mixture was resolved by gel electrophoresis, theapproximately 1.7 kb DNA fragment comprising the α-farnesene synthasecoding sequence was gel extracted, and the isolated DNA fragment wasligated into the NcoI SacI restriction enzyme site of the pTrc99Avector, yielding expression plasmid pTrc99A-FSA (see FIG. 6 for aplasmid map). The DNA fragments for GTS, APS, and TS were designed to beflanked by a leader XmaI restriction enzyme site and a terminal XbaIrestriction enzyme site, and were cloned into compatible restrictionenzyme sites of a cloning vector such as a standard pUC or pACYC originvector, from which they could be liberated again by digesting tocompletion the DNA synthesis construct using XbaI and XmaI restrictionenzymes, resolving the reaction mixture by gel electrophoresis, and gelextracting the 1.7 to 1.9 terpene synthase encoding DNA fragment. Theisolated DNA fragments were ligated into the XmaI XbaI restrictionenzyme site of vector pTrc99A (Amman et al., Gene 40:183-190 (1985)),yielding plasmids pTrc99A-GTS, pTrc99A-APS, or pTrc99A-TS (see FIG. 6for plasmid maps).

Expression plasmids pRS425-FSA and pRS425-FSB were generated byinserting a nucleotide sequence encoding an α-farnesene synthase (“FSA”)or a β-farnesene synthase (“FSB”), respectively, into the pRS425-Gallvector (Mumberg et. al. (1994) Nucl. Acids. Res. 22(25): 5767-5768). Thenucleotide sequence inserts were generated synthetically, using as atemplate for example the coding sequence of the α-farnesene synthasegene of Picea abies (GenBank accession number AY473627, REGION: 24 . . .1766) or of the β-farnesene synthase gene of Artemisia annua (GenBankaccession number AY835398), codon-optimized for expression inSaccharomyces cerevisiae. The synthetically generated nucleotidesequence was flanked by a 5′ BamHI site and a 3′ XhoI site, and couldthus be cloned into compatible restriction enzyme sites of a cloningvector such as a standard pUC or pACYC origin vector. The syntheticallygenerated nucleotide sequence was isolated by digesting to completionthe DNA synthesis construct using BamHI and XhoI restriction enzymes.The reaction mixture was resolved by gel electrophoresis, theapproximately 1.7 kb DNA fragment comprising the α-farnesene synthase orβ-farnesene synthase coding sequence was gel extracted, and the isolatedDNA fragment was ligated into the BamHI XhoI restriction enzyme site ofthe pRS425-Gall vector, yielding expression plasmid pRS425-FSA orpRS425-FSB, respectively.

Expression plasmids pTrc99A-LLS, pTrc99A-LMS, pTrc99A-BPS, pTrc99A-PHS,pTrc99A-CS, and pTrc99A-SS are generated by inserting a nucleotidesequence encoding a linalool synthase (“LLS”), limonene synthase(“LMS”), β-pinene synthase (“BPS”), β-phellandrene (“PHS”), carenesynthase (“CS”), or sabinine synthase (“SS”) into the pTrc99A vector.The nucleotide sequence inserts are generated synthetically, using as atemplate for example the coding sequence of the linalool synthase geneof Artemisia annua (GenBank accession number AF154124, REGION: 13.1764),the coding sequence of the limonene synthase gene of Abies grandis(GenBank accession number AF006193 REGION: 73.1986), the coding sequenceof the β-pinene synthase of Artemisia annua (GenBank accession numberAF276072 REGION: 1.1749), the coding sequence of the β-phellandrenesynthase gene of Abies grandis (GenBank accession number AF139205REGION: 34.1926), the coding sequence of the carene synthase gene ofSalvia stenophylla (GenBank accession number AF527416 REGION: 78.1871),or the coding sequence of the sabinene synthase gene of Salviaofficinalis (GenBank accession number AF051901 REGION: 26.1798). Thenucleotide sequences encoding the 3-pinene, sabinine, and β-phellandrenesynthases are flanked by a leader XmaI restriction enzyme site and aterminal XbaI restriction enzyme site, the nucleotide sequences encodingthe linalool and carene synthases are flanked by a leader NcoIrestriction enzyme site and a terminal XmaI restriction enzyme site, andthe nucleotide sequence encoding the limonene synthase is flanked by aleader NcoI restriction enzyme site and a terminal PstI restrictionenzyme site. The DNA synthesis constructs are digested to completingusing XmaI and XbaI (for the β-pinene, sabinine, and β-phellandrenesynthase constructs), NcoI and XmaI restriction enzymes (for thelinalool and careen synthase constructs), or XbaI and PstI restrictionenzymes (for the limonene synthase construct). The reaction mixtures areresolved by gel electrophoresis, the approximately 1.7 to 1.9 kb DNAfragments are gel extracted, and the isolated DNA fragments are ligatedinto the XmaI XbaI restriction enzyme site (for the β-pinene, sabinine,and β-phellandrene synthase inserts), the NcoI XmaI restriction enzymesite (for the linalool and carene synthase inserts), or the XbaI PstIrestriction enzyme site (for the limonene synthase insert) of thepTrc99A vector, yielding expression plasmids pTrc99A-LLS, pTrc99A-LMS,pTrc99A-BPS, pTrc99A-PHS, pTrc99A-CS, and pTrc99A-SS (see FIG. 6 forplasmid maps).

Example 6

This example describes the generation of Escherichia coli host strainsuseful in the invention.

As detailed in Table 1, the host strains were created by transformingchemically competent Escherichia coli parent cells with one or moreexpression plasmids of Example 1 through 5.

TABLE 1 E. coli host strains E. coli Parent Expression Antibiotic HostStrain Strain Plasmids Selection B32 DH1 pMevT 100 ug/mL B292 B pMBIScarbenicillin B210 DP pTrc99A-ADS  5 ug/mL tetracycline  34 ug/mLchloramphenicol B153 DH1 pAM97 100 ug/mL B282 DP pTrc99A-ADScarbenicillin  34 ug/mL chloramphenicol B255 DH1 pAM128 100 ug/mL B256DP pAM113 carbenicillin  34 ug/mL chloramphenicol B86 DH1 pAM52  50ug/mL kanamycin pMBIS 100 ug/mL pTrc99A-ADS carbenicillin B61 DH1 pAM25 5 ug/mL pBBR1MCS-3 tetracycline pTrc99A B62 pAM34 pBBR1MCS-3 pTrc99AB003 DH10B pTrc99A-ADS 100 μg/ml carbenicillin B617 pAM408 100 ug/mLpTrc99A-ADS carbenicillin  50 ug/mL kanamycin B618 pAM424 100 ug/mLpTrc99A-ADS carbenicillin  35 μg/ml chloramphenicol B619 pAM408 100μg/ml pAM424 carbenicillin pTrc99A-ADS  50 μg/ml kanamycin  35 μg/mlchloramphenicol B650 DH10B pAM373 100 μg/ml carbenicillin B651 pAM408100 μg/ml pAM373 carbenicillin  50 μg/ml kanamycin B652 pAM424 100 μg/mlpAM373 carbenicillin  35 μg/ml chloramphenicol B653 pAM408 100 μg/mlpAM424 carbenicillin pAM373  50 μg/ml kanamycin  35 μg/mlchloramphenicol B286 DH1 pAM97-MevB 100 ug/mL pC9 carbenicillin B287pAM97-MevB  34 ug/mL pnudF-H chloramphenicol. B288 pAM97-MevB pyhfR B291pAM97-MBI pyhfR B592 DH1 pMevT 100 ug/mL pMBIS carbenicillin pTrc99A-FSA 34 ug/mL B552 pMevT chloramphenicol pMBIS  5 ug/mL pAM373 tetracyclineExample 21 host cell pMevT (production of GTS, pMBIS-gpps APS, TS)pTrc99A-GTS or -APS or -TS Example 21 host cell pMevT 100 ug/mL(production of LLS, pMBIS-gpps carbenicillin LMS, BPS, PHS, CS,pTrc99A-LLS or  34 ug/mL SS) -LMS or -BPS or chloramphenicol -PHS or -CSor  5 ug/mL -SS tetracycline

Host cell transformants were selected on Luria Bertoni (LB) agarcontaining antibiotics as detailed in Table 1. Single colonies weretransferred from LB agar to culture tubes containing 5 mL of LB liquidmedium and antibiotics. B003, B617, B618, B619, B650, B651, B652, andB653 host cell transformants were incubated at 30° C. on a rotary shakerat 250 rpm for 30 hours. All other host cell transformants wereincubated at 37° C. on a rotary shaker at 250 rpm until growth reachedstationary phase. The cells were adapted to minimal media by passagingthem through 4 to 5 successive rounds of M9-MOPS media containing 0.8%glucose and antibiotics (see Table 2 for the composition of the M9-MOPSmedium). The cells were stored at −80° C. in cryo-vials in 1 mL stockaliquots made up of 400 uL sterile 50% glycerol and 600 uL liquidculture.

TABLE 2 Composition of M9-MOPS Culture Medium Component Quantity (per L)Na₂HPO₄7H₂O 12.8 g KH₂PO₄ 3 g NaCl 0.5 g NH₄Cl 1 g MgSO₄ 2 mmol CaCl₂0.1 mmol Thiamine 0.1 ug MOPS buffer pH 7.4 100 mmol (NH₃)6Mo7O24 4H2O3.7 ug H₃BO₄ 25 ug CoCl₂ 7.1 ug CuSO₄ 2.4 ug MnCl₂ 16 ug ZnSO₄ 2.9 ugFeSO₄ 0.28 mg

Example 7

This example demonstrates expression plasmid stability in the absence ofantibiotics in an Escherichia coli host strain that harbors anexpression plasmid comprising the RK2 plasmid replication, segregation,and maintenance system.

A seed culture of host strain B255 was established by adding a stockaliquot of the strain to a 125 mL flask containing 40 mL M9-MOPS, 2%glucose, 0.5% yeast extract, and antibiotics as detailed in Table 1, andby growing the culture overnight.

The seed culture was used to inoculate at an initial OD₆₀₀ ofapproximately 0.05, two 250 mL flasks each containing 40 mL M9-MOPSmedium, 2% glucose, and 0.5% yeast extract. Culture #1 also contained100 ug/mL carbenicillin and 34 ug/mL chloramphenicol. Culture #2 did notreceive any antibiotics. Both cultures were incubated at 37° C. on arotary shaker at 250 rpm until they reached an OD₆₀₀ of approximately0.2, at which point the production of amorpha-4,11-diene in the hostcells was induced by adding 40 uL of 1M IPTG to the culture medium. Atthe time of induction, the cultures were overlain with 8 mL of anorganic overlay to capture the amorpha-4,11-diene. Samples were takenperiodically for a total of 72 hours. Production of amorpha-4,11-dieneby the host strain in the 2 cultures was confirmed by GC/MS as describedin Example 10.

To assess plasmid stability in the two cell cultures, a sample of eachculture was removed at 72 hours and streaked onto a LB agar plate (noantibiotics). After overnight incubation at 37° C., 50 individualcolonies derived from each culture were replica-plated onto a LBagar-plus-antibiotics (34 ug/mL chloramphenicol, 100 ug/mLcarbenicillin) plate and a LB agar-minus-antibiotics (no antibiotic)plate. After another overnight incubation at 37° C., the LBagar-plus-antibiotics and the LB agar-minus-antibiotics plate were eachfound to contain approximately 50 colonies, indicating that plasmidretention both in the presence and in the absence of antibiotics in theculture medium had been approximately 100%.

Example 8

This example demonstrates increased specific activity and stability ofthe Enterococcus faecalis HMGR compared to the Saccharomyces cerevisiaetHMGR in an Escherichia coli host strain.

Seed cultures of host strains B61 and B62 were established by adding astock aliquot of each strain to 125 mL flasks containing 20 mL M9-MOPSmedium, 0.8% % glucose, and antibiotics as detailed in Table 5, and bygrowing the cultures to saturation. The seed cultures were diluted 1:100into 140 mL of fresh medium in a 500 mL flask, and grown again to anOD₅₅₀ of approximately 0.1, at which point production ofamorpha-4,11-diene was induced by adding 140 uL 1 M IPTG to eachculture. At 4, 12, 20, 28, 36, and 49 hours post-induction, samples wereremoved from each culture, and cells were pelleted by centrifugation.The cell pellets were snap frozen on dry ice, and then stored at −80° C.

To conduct enzyme assays, cell pellets were thawed on ice, and thenlysed using Bugbuster (Novagen, Madison, Wis.) containing proteaseinhibtor mix #3 (Calbiochem, San Diego, Calif.), benzonase (20 μL oer5mL bugbuster; Novagen, Madison, Wis.), and lysozyme (30 ug/mL). Enzymeactivity of the Saccharomyces cerevisiae tHMGR was assayed in 50 mM TrisHCl (pH7.5), 0.2 mM NADPH (Sigma, St. Louis, Mo.), and 0.3 mMDL-3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) sodium salt (Sigma,St. Louis, Mo.). The assay was started by adding cell lysate, and thedisappearance of NADPH was monitored by absorbance at 340 nM. To accountfor non-specific disappearance of NADPH, results obtained in a controlassay lacking HMG-CoA were subtracted from results obtained in testsamples. Enzyme activity of the Enterococcus faecalis HMGR was measuredsimilarly except that the assay buffer contained 100 mM potassiumphosphate buffer (pH6.5), 0.4 mM NADPH, 1.0 mM EDTA, and 100 mM KCl.

Protein assays were done by the method of Bradford ((1976) Anal Biochem.72:248-254). Specific activities were calculated as Δnmol NADPH/min/mgprotein.

As shown in FIG. 8, the Enterococcus faecalis HMGR exhibited higherspecific activity and increased stability compared to the Saccharomycescerevisiae tHMGR.

Example 9

This example describes the calibration of OD₆₀₀ with dry cell weight(“DCW”).

To obtain the relationship between DCW and OD600, a representativestrain, B32, was grown in high cell density processes similar to thosedescribed in Examples 10-14. Samples were taken throughout the runs, andthe OD₆₀₀ and DCW were measured for each sample. To determine the DCW,the cells were pelleted and the supernatant discarded. The cell pelletwas washed once with water, and was then dried in an oven at 80° C. forat least 3 days. The tubes containing cell pellets were weighed, theweight of the tube was subtracted from the measured weights, and theremaining weight was divided by the initial volume of each sample(0.0015 L) to obtain the DCW.

FIG. 9 shows the relationship between DCW and OD₆₀₀ measured in theseexperiments.

Example 10

This example demonstrates increased production of amorpha-4,11-diene inEscherichia coli host strains expressing the Staphylococcus aureus HMGRand HMGS compared to host strains expressing the Saccharomycescerevisiae tHMGR and HMGS.

Seed cultures of host strains B32, B153, B210, B282, B292, B86, B255,and B256 were established by adding a stock aliquot of each strain toseparate 125 mL flasks containing 25 mL M9-MOPS medium, 0.8% glucose,and antibiotics as detailed in Table 1, and by growing the culturesovernight.

The seed cultures were used to inoculate at an initial OD₆₀₀ ofapproximately 0.05 separate 250 mL flasks containing 40 mL M9-MOPSmedium, 2% glucose, and antibiotics. The cultures were incubated at 30°C. on a rotary shaker at 250 rpm until they reached an OD₆₀₀ ofapproximately 0.2, at which point the production of amorpha-4,11-dienein the host cells was induced by adding 40 uL of 1M IPTG to the culturemedium. The cultures were overlain with 8 mL of an organic overlay(e.g., dodecane, methyl oleate or isopropyl myristate). Samples of theorganic overlay layer and the broth were taken once a day for 72 hours.Broth samples were used to measure the OD₆₀₀. Amorpha-4,11-dieneconcentration was measured by transferring 5 uL of the organic overlaylayer to a clean glass vial containing 500 uL ethyl acetate spiked withbeta- or trans-caryophyllene as an internal standard.

The organic overlay/ethyl acetate samples were analyzed on aHewlett-Packard 6890 gas chromatograph/mass spectrometer (GC/MS) byscanning only for two ions, the molecular ion (204 m/z) and the 189 m/zion, as described in Martin et al. (2001) Biotechnol. Bioeng.75:497-503. To expedite run times, the temperature program and columnmatrix was modified to achieve optimal peak resolution and the shortestoverall runtime. A 1 uL sample was separated on the GC using a DB-XLBcolumn (available from Agilent Technologies, Inc., Palo Alto, Calif.)and helium carrier gas. The temperature program for the analysis was asfollows: 100° C. for 0.75 minutes, increasing temperature at 60°C./minute to a temperature of 300° C., and a hold at 300° C. for 0.5minutes. The resolved samples were analyzed by a Hewlett-Packard model5973 mass-selective detector that monitored ions 189 and 204 m/z.Previous mass spectra demonstrated that the amorpha-4,11-diene synthaseproduct was amorpha-4,11-diene, and that amorpha-4,11-diene had aretention time of 3.7 minutes using this GC protocol. Beta- ortrans-caryophyllene was used as an internal standard for quantitation.Amorpha-4,11-diene titer was calculated using the ratio of internalstandard to amorpha-4,11-diene peak areas based upon a quantitativecalibration curve of purified amorpha-4,11-diene (0.63-10 mg/L ofKJF17-109-3) in caryophyllene-spiked ethyl acetate.

As shown in FIGS. 10A and 10B, strains B153 and B282, which expressedthe Staphylococcus aureus HMGR and HMGS, produced elevated levels ofamorpha-4,11-diene compared to strains B32, B210, B255, B256, and B292,which expressed the Saccharomyces cerevisiae tHMGR and HMGS.

Example 11

This example demonstrates increased production of amorpha-4,11-diene byan

Escherichia coli host strain grown at suboptimal temperature.

A seed culture of host strain B32 was established by adding 0.5 mL of astock aliquot of the strain to a 250 mL flask containing 50 mL M9-MOPSmedium and antibiotics as detailed in Table 1, and by growing theculture overnight at 37° C. on a rotary shaker at 250 rpm.

The seed culture was used to inoculate at an initial OD₆₀₀ ofapproximately 0.05 four 250 mL flasks, each containing 40 mL fermentorbatch medium (see Table 6 for medium composition), 100 mM MOPS bufferpH7.1, and antibiotics. The cultures were incubated on a rotary shakerat 250 rpm at either 30° C. or 37° C. until they reached an OD₆₀₀ of0.18 to 0.22, at which point the production of amorpha-4,11-diene in thehost cells was induced by adding 40 uL of 1M IPTG to the culture medium.At the time of induction, the cultures were overlain with 8 mL of anorganic overlay to capture the amorpha-4,11-diene. Samples were takenonce a day, and analyzed as described in Example 10.

As shown in FIGS. 11A and 11B, fermentation at 30° C. did not affectcell growth, but led to an approximately 50% increase in the specificproduction of amorpha-4,11-diene by the Escherichia coli host strain.

Example 12

This example demonstrates increased production of amorpha-4,11-diene byan Escherichia coli host strain grown under restricted carbon sourceconditions.

A seed culture of host strain B32 for fermentation runs 050608-1 and050629-1 was established by adding 0.25 uL of a stock aliquot of thestrain to a 250 mL flask containing 50 mL M9-MOPS medium and antibioticsas detailed in Table 1, and by incubating the culture at 37° C. on arotary shaker at 250 rpm until it reached an OD₆₀₀ of 1 to 2.

A seed culture of host strain B32 for fermentation run 060403-3 wasestablished by adding a stock aliquot of the strain to a 250 mL flaskcontaining 50 mL M9-MOPS medium and antibiotics as detailed in Table 1,and by incubating the culture overnight at 37° C. on a rotary shaker at250 rpm. The seed culture was used to inoculate at an initial OD₆₀₀ ofapproximately 1 a 250 mL flask containing 40 mL M9-MOPS medium andantibiotics, and the culture was again incubated at 37° C. on a rotaryshaker at 250 rpm until it reached an OD₆₀₀ of 3 to 5.

For all fermentation processes, the KH₂PO₄, K₂HPO₄ 3H₂O, EDTA, citricacid, and (NH₄)₂SO₄ were heat sterilized in the bioreactor (2 L ApplikonBioconsole ADI 1025s with ADI 1010 controllers, Applikon Biotechnology,Foster City, Calif.). The remaining media components were filtersterilized as stock solutions and injected through the headplate. Table3 shows the final media composition for fermentation runs 050608-1 and050629-1. Table 4 shows the final media composition for fermentation run060403-3. The starting volume for run 050608-1 was 0.8 L, the startingvolume for 050629-1 was 1.2 L and the starting volume for 060403-3 was 1L. All runs were inoculated by injecting 50 mL of the seed culturethrough the headplate.

TABLE 3 Composition of Fermentation Medium of Fermentation Runs 050608-1and 050629-1 Component Batch Medium (per L) Feed Solution (per L)Glucose 5 g 590-650 g KH₂PO₄ 4.2 g — K₂HPO₄ 3H₂O 15.7 g — Citric acid1.7 g — (NH₄)₂SO₄ 2 g — MgSO₄ 7H₂O 1.2 g 12 g EDTA 8.4 mg 13 g CoCl₂6H₂O 0.25 mg 0.4 mg MnCl₂ 4H₂O 1.5 mg 2.35 mg CuCl₂ 2H₂O 0.15 mg 0.25 mgH₃BO₄ 0.3 mg 0.5 mg Na₂MoO₄ 2H₂O 0.25 mg 0.4 mg Zn(CH₃COO)₂ 2H₂O 1.3 mg1.6 mg Fe(III)citrate 10.0 mg 4.0 mg hydrate Thiamine HCl 4.5 mg —Carbenicillin 100 ug 100 ug Tetracycline 5 ug 5 ug Chloramphenicol 34 ug34 ug

TABLE 4 Composition of Fermentation Medium of Fermentation Run 060403-3Batch medium (per Feed solution Component L) (per L) Glucose 15 g 650 gKH₂PO₄ 4.2 g — K₂HPO₄ 3H₂O 15.7 g — Citric acid 1.7 g — (NH₄)2SO₄ 2 g —MgSO₄ 7H₂O 1.2 g 12 g EDTA 8.4 mg 13 mg CoCl₂ 6H₂O 2.5 mg 4 mg MnCl₂4H₂O 15 mg 23.5 mg CuCl₂ 2H₂O 1.5 mg 2.5 mg H₃BO₄ 3 mg 5 mg Na₂MoO₄ 2H₂O2.5 mg 4 mg Zn(CH₃COO)₂ 2H₂O 13 mg 16 mg Fe(III)citrate hydrate 100 mg40 mg Thiamine HCl 4.5 mg — Carbenicillin 100 ug 100 ug Tetracycline 5ug 5 ug Chloramphenicol 34 ug 34 ug

For fermentation run 050608-1 (excess carbon), the feed was initiated atinduction, and feed rates were adjusted manually to provide glucose inthe concentrations shown in FIG. 12C. For fermentation run 050629-1(carbon-restricted), the feed was delivered to the fermentor accordingto the protocol shown in Table 5. For fermentation run 060403-3 (lowestcarbon), the feed was started automatically when the initial glucosebolus (15 g) was exhausted and the dissolved oxygen spiked. Up to amaximum of 27.6 g/hr, the rate of the feed was calculated according tothe following equation:m _(s)(t)=S(t ₀)μe ^(μ(t-t) ⁰ ⁾μ=0.12S(t ₀)=15 gwherein t₀ is the time at which the initial glucose was depleted. Uponreaching the maximum rate, the glucose feed was restricted to a rate of9.5 g/hr, and held constant at this rate for the remainder of the run.

TABLE 5 Feed Protocol for Fermentation Run 050629-1 Glucose Feed RateRun Time (hours) (g/hr) 0 0 7 0.37 10 0.74 12 1.11 14 1.48 16 2.22 182.96 20 3.69 22 4.80 24 5.91 31 7.39 33 5.54 47 3.69

Runs 050608-1 and 050629-1 were carried out at 37° C. Airflow in thebioreactor was set at 1-2 L/min; pH was maintained at 7 using ammoniumhydroxide and/or sodium hydroxide; initial agitation was 500-600 rpm;foam was controlled with antifoam B (Sigma-Aldich, St. Louis, Mo.); thedissolved oxygen levels were maintained above 30% using an agitationcascade. After 5-6 hours of cultivation, production ofamorpha-4,11-diene by the host cells was induced by adding 0.8 mL of 1 MIPTG to run 050608-1 and 1.2 mL IPTG to run 050629-1. Upon induction,the culture temperature was reduced to 30° C.

Run 060403-3 was carried out at 30° C. Airflow in the bioreactor was setat 1-2 L/min; pH was maintained at 7 using ammonia hydroxide. Dissolvedoxygen was maintained above 30% by an agitation cascade and oxygenenrichment. At an OD₆₀₀ of approximately 28 (19 hours afterinoculation), production of amorpha-4,11-diene by the host cells wasinduced by adding 1 mL 1 M IPTG.

Amorpha-4,11-diene was captured and extracted according to two differentprotocols. For runs 050608-1 and 050629-1, volatile amorpha-4,11-dienepresent in the off-gas was captured by venting the off-gas through agas-washer containing 200 mL heptanol. The heptanol was then dilutedinto ethyl acetate until the amorpha-4,11-diene concentration in thesample was between 0.63 mg/L and 20 mg/L. For run 060403-3,amorpha-4,11-diene was captured in the bioreactor by adding 200 mL of anorganic overlay to the fermentor at the time of induction. Productconcentration was measured by combining 25 uL broth plus organic overlaywith 975 uL acetonitrile, shaking the sample at maximum speed on aFisher Vortex Genie 2™ mixer (Scientific Industries, Inc., Bohemia, NY)for at least 3 minutes, removing cells from the sample bycentrifugation, and diluting the acetonitrile solution into ethylacetate until the amorpha-4.11-diene concentration in the sample wasbetween 0.63 and 20 mg/L. The ethyl acetate samples were analyzed byGC/MS as described in Example 10.

As shown in FIGS. 12A and 12B, fermentation run 050608-1 (excess carbon)resulted in low maximum cell densities and low production ofamorpha-4,11-diene, respectively, correlating, at least in part, to therelatively rapid increase in acetate levels (FIG. 12D). In comparison,fermentation run 050629-1 (carbon-restricted) resulted in increasedproduction of amorpha-4,11-diene (FIG. 12B), and delayed the onset ofacetate production. These results are consistent with the hypothesisthat excess glucose feeds lead to rapid acetate production and earlycell death.

Further glucose restriction as achieved by fermentation run 060403-3(lowest carbon) resulted in low acetate production for over 100 hours(FIG. 12D), and significantly higher maximum cell density andamorpha-4,11-diene production (FIGS. 12A and 12B).

Example 13

This example demonstrates increased amorpha-4,11-diene production by anEscherichia coli host strain grown under restricted carbon sourceconditions and at suboptimal temperature.

A seed culture of host strain B153 was established by adding a stockaliquot of the strain to a 250 mL flask containing 50 mL M9-MOPS mediumand antibiotics as detailed in Table 1, and growing the culture at 37°C. on a rotary shaker at 250 rpm to an OD₆₀₀ of 3.5 to 4.5.

2 L bioreactors (Biocontroller ADI 1010 with Bioconsole ADI 1025,Applikon Biotechnology, Foster City, Calif.) were set up and run in thesame way as described in Example 12 for run 060403-3, except that strainand induction time were varied.

Production of amorpha-4,11-diene in the host cells was induced by adding1 mL of 1 M IPTG to the culture medium. In the fermentation run shown inFIG. 13A, amorpha-4,11-diene synthesis was induced at an OD₆₀₀ ofapproximately 2, while the fermentor still contained excess glucose. Inthe fermentation run shown in FIG. 13B, amorpha-4,11-diene synthesis wasinduced at an OD₆₀₀ of approximately 33, which was after theglucose-restricted feed had started.

Amorpha-4,11-diene was captured and extracted according to two differentprotocols. For the fermentation run shown in FIG. 13A, volatileamorpha-4,11-diene present in the off-gas was captured by venting theoff-gas through a gas-washer containing 200 mL heptanol. The heptanolwas then diluted into ethyl acetate until the amorpha-4,11-dieneconcentration in the sample was between 0.63 and 20 mg/L. For thefermentation run shown in FIG. 13B, amorpha-4,11-diene was captured byadding 200 mL of an organic overlay to the fermentor at the time ofinduction.

Amorpha-4,11-diene was extracted from the culture medium by combining 25uL broth with 975 uL acetonitrile, shaking the sample at maximum speedon a Fisher Vortex Genie 2™ mixer (Scientific Industries, Inc., Bohemia,N.Y.) for at least 3 minutes, removing cells from the sample bycentrifugation, and diluting the acetonitrile solution into ethylacetate until the amorpha-4.11-diene concentration in the sample wasbetween 0.63 and 20 mg/L. The ethyl acetate samples were analyzed byGC/MS as described in Example 10. For the fermentation run shown in FIG.13A, the total amount of amorpha-4,11-dien was derived by combining theamounts present in the off-gas and in the culture medium, and dividingthe total by the fermentor volume.

The fermentation shown in FIG. 13A reached a maximal OD₆₀₀ of 93 and amaximal amorpha-4,11-diene concentration of 3.2 g/L. In contrast, thefermentation shown in FIG. 13B reached a maximal OD₆₀₀ of 245 and amaximal amorpha-4,11-diene concentration of 15 g/L. A likely explanationfor the differences in culture growth and amorpha-4,11-diene productionlevels observed in the two cultures is that in the fermentation runshown in FIG. 13A amorpha-4,11-diene production was induced before theexcess glucose was consumed, and that the unrestricted availability ofglucose caused cell death by enabling the build-up of toxic levels ofintermediates of the mevalonate pathway. In the fermentation run shownin FIG. 13B, induction occurred after glucose delivery was restricted,which prevented the build-up of pathway intermediates, leading to highercell density and amorpha-4,11-diene production levels.

Example 14

This example demonstrates increased amorpha-4,11-diene production by anEscherichia coli host strain grown under restricted carbon and nitrogensource conditions and at suboptimal temperature.

A seed culture of host strain B86 was established by adding a stockaliquot of the strain to a 250 mL flask containing 50 mL M9-MOPS mediumand antibiotics as detailed in Table 1. The culture was grown overnightat 37° C. on a rotary shaker at 250 rpm, sub-cultured the followingmorning into the same medium at an OD₆₀₀ of approximately 1, and grownagain at 37° C. and 250 rpm to an OD₆₀₀ of 3 to 5.

Four 2 L bioreactors (Biocontroller ADI 1010 with Bioconsole ADI 1025,Applikon Biotechnology, Foster City, Calif.) were set up and run in thesame way as described in Example 12 for run 060403-3, except that thenitrogen restricted runs did not contain ammonia sulfate in the feed.

An exponential glucose feed with a 6 hour doubling time was initiatedautomatically when the initial glucose bolus (15 g) was exhausted andthe dissolved oxygen spiked. Up to a maximum of 30.4 g/hr, the rate ofthe feed was calculated according to the following equation:m _(s)(t)=S ₀ μe ^(μ(t-t) ⁰ ⁾μ=0.12 min⁻¹S ₀=15 gwherein μ is the specific growth rate, and t₀ is the time at which theinitial glucose bolus was depleted. Upon reaching the maximum rate, theglucose feed was reduced to a rate of 11.4 g/hr, and held constant atthis rate for the remainder of the run. In fermentation runs 060710-4,060724-5, and 060619-5 (carbon- and nitrogen-restricted), the glucosefeed was further reduced when ammonia restriction lead to glucoseaccumulation in the medium.

Fermentation was carried out at the reduced temperature of 30° C.Airflow in the bioreactor was set at 1 vvm; initial agitation was at 700rpm; foam was controlled with antifoam B (Sigma-Aldich, St. Louis, Mo.);and dissolved oxygen tension was controlled at 40% using an agitationcascade (700-1,200 rpm) and oxygen enrichment. In fermentation run060327-3 (carbon-restricted), the pH was maintained at 7 using 20%NH₄OH; in fermentation runs 060710-4, 060724-5, and 060619-5 (carbon-and nitrogen-restricted), pH was maintained at 7 initially using 20%NH₄OH, and starting at 72 hours using a 50/50 mixture of 2.5 N NaOH and10 N NH₄OH, to further restrict the amount of ammonia going into thefermentor.

Production of amorpha-4,11-diene in the host cells was induced at anOD₆₀₀ of approximately 30 by adding 1 mL of 1 M IPTG to the culturemedium.

Amorpha-4,11-diene was captured by overlaying the medium with 10% (v/v)of an organic overlay. Amorpha-4,11-diene was then extracted bycombining 25 uL of broth with 975 uL methanol, shaking the sample atmaximum speed on a Fisher Vortex Genie 2™ mixer (Scientific Industries,Inc., Bohemia, N.Y.) for at least 15 minutes, removing cells from thesample by centrifugation, and adding 10 uL of the methanol solution to990 uL ethyl acetate containing 10 uL/L trans-caryophylene.

Samples were analyzed by GC/MS as described in Example 10.

FIGS. 14A-14E show data from fermentation run 060327-3(carbon-restricted). The fermentation produced a maximum concentrationof amorpha-4,11-diene of 16 g/L (FIG. 14A). The maximum volumetricproductivity of the host strain was more than 200 mg/L/hour (FIG. 14B).The maximum specific productivity of the host strain was >2mg/L/hour/OD₆₀₀ (FIG. 14C). The concentration of ammonia in the culturemedium was about 30 mM at the start of the fermentation run, rose toabout 76 mM upon addition of the feed solution during the exponentialgrowth phase, and remained above 60 mM for the remainder of the run(FIG. 14D). The maximum OD₆₀₀ reached was about 290 (FIG. 14D),corresponding to 116 g DCW/L. The concentration of glucose in theculture medium dropped from 15 g/L to below 1 g/L in less than 20 hours,and remained low (FIG. 14E). Acetate levels were low throughout thefermentation (FIG. 14E).

FIGS. 15A-E show data from fermentation runs 060710-4, 060724-5, and060619-5 (carbon- and nitrogen-restricted). The fermentations produced amaximum concentration of amorpha-4,11-diene from about 20 g/L to 30 g/L(FIG. 15A). The maximum volumetric productivity of the host strain wasmore than 400 mg/L/hour in all three fermentation runs (FIG. 15B), whichis significantly higher than the maximum volumetric productivityobtained in the nitrogen unrestricted fermentation (FIG. 14B). Themaximum specific productivity of the host strain was >2 mg/L/hour/OD₆₀₀for all runs, and remained high throughout the runs (FIG. 15C). Theconcentration of ammonia in the culture medium was about 35 mM to 50 mMat the start of the fermentation runs, dropped upon addition of the feedsolution during exponential growth, and remained below 10 mM for theremainder of the run (FIG. 15D). (The lowered ammonia levels compared tofermentation run 060327-3 (FIG. 14D) are due to the lack of ammonia inthe feed solution and reduced ammonia in the base used to maintain thepH. Fermentation runs 060710-4 and 060619-5 showed a spike in ammoniaconcentration at the end of the runs, but the spikes occurred after thebulk of the production of amorpha-4,11-diene) The maximum OD₆₀₀ reachedwas 170 to 220 (FIG. 15D), corresponding to 68 g to 88 g DCW/L. Theconcentration of glucose in the culture medium dropped from 15 g/L tobelow 1 g/L in less than 20 hours, and remained low (FIG. 15E). Acetatelevels were low throughout the fermentation runs (FIG. 15E).

Example 15

This example describes the production of amorpha-4,11-diene via the DXPpathway in an Escherichia coli host strain.

Seed cultures of host strains B003, B617, B618, and B619 wereestablished by adding a stock aliquot of each strain to separate 125 mLflasks containing 25 mL M9-MOPS and antibiotics as detailed in Table 1,and by growing the cultures overnight.

The seed cultures were used to inoculate at an initial OD₆₀₀ ofapproximately 0.05, separate 250 mL flasks containing 40 mL M9-MOPSmedium, 45 ug/mL thiamine, micronutrients, 1.00E-5 mol/L FeSO4, 0.1 MMOPS, 0.5% yeast extract, 20 g/L of D-glucose, and antibiotics. Cultureswere incubated at 30° C. in a humidified incubating shaker at 250 rpmuntil they reached an OD₆₀₀ of 0.2 to 0.3, at which point the productionof amorpha-4,11-diene in the host cells was induced by adding 40 uL of1M IPTG to the culture medium.

At the time of induction, the cultures were overlain with 8 mL of anorganic overlay to capture the amorpha-4,11-diene. Samples were taken atvarious time points, and amorpha-4,11-diene was extracted and analyzedby GC/MS as described in Example 10. Experiments were performed using 2independent clones of each host strain, and results were averaged.Deviation between samples was found to be less than 10%.

As shown in FIG. 16, Escherichia coli host strain B619, which comprisesnucleotide sequences encoding enzymes of the full engineered DXPpathway, produced approximately 45 mg/g DCW amorpha-4,11-diene.

Example 16

This example describes the production of 3-methyl-but-3-en-1-ol and3-methyl-but-2-en-1-ol in Escherichia coli host strains.

Seed cultures of host strains B286, B287, B288, and B291 wereestablished by streaking out a stock aliquot of each strain on LB agarcontaining antibiotics as detailed in Table 1. Three independentcolonies were picked for each strain, and each colony was inoculatedinto 7 mL of LB media containing antibiotics. The cultures were grownovernight at 37° C. on a rotary shaker at 250 rpm until late exponentialphase. The cultures were then inoculated at an OD₆₀₀ of approximately0.05, into a 250 mL flask containing 40 ml of M9-MOPS, 2% glucose, 0.5%yeast extract, and antibiotics. The cultures were grown overnight at 37°C. on a rotary shaker at 250 rpm until they reached an OD₆₀₀ ofapproximately 0.2, at which point they were induced by adding 40 uL of 1M IPTG. The cultures were grown for 72 hours at 30° C. on a rotaryshaker at 250 rpm. One to two times per day, the OD₆₀₀ of each culturewas measured, and a 700 uL sample was removed. To extract the3-methyl-but-3-en-1-ol and 3-methyl-but-2-en-1-ol from the culturebroth, 600 uL of ethyl acetate was added to 300 uL of each removedsample. The sample was then vortexed for 15 minutes, and 400 uL of theupper ethyl acetate phase was transferred to a clean glass vial foranalysis.

The samples were analyzed on a Hewlett-Packard 6890 gaschromatograph/mass spectrometer (GC/MS). A 1 uL sample was separated onthe GC using a DB-5 column (Agilent Technologies, Inc., Palo Alto,Calif.) and helium carrier gas. The temperature program for the analysiswas as follows: 60° C. for 3 minutes, increasing temperature at 60°C./minute to a temperature of 300° C., and a hold at 300° C. for 2minutes. The total run time was 9 minutes. The resolved samples wereanalyzed by a Hewlett-Packard model 5973 mass selective detector.Previous mass spectra demonstrated that 3-methyl-3-buten-1-ol and3-methyl-2-buten-1-ol have a retention time of 2.067 minutes using thisGC protocol. To focus detection on 3-methyl-but-3-en-1-ol and3-methyl-but-2-en-1-ol, a selective-ion-monitoring method was employedthat monitors only ions 56 and 68 in 3-methyl-but-3-en-1-ol and3-methyl-but-2-en-1-ol.

Example 17

This example describes the production of amorpha-4,11-diene by aSaccharomyces cerevisiae host strain.

The generation of host strain EPY224 is described in Ro et al. (Nature440: 940-943; 2006) and in PCT Patent Publication WO2007/005604. Hoststrain EPY224 was cured of expression plasmid pRS425ADS by growth in YPDmedium (Methods in Yeast Genetics: A Cold Spring Harbor LaboratoryCourse Manual, 2005 ed., ISBN 0-87969-728-8), plating for singlecolonies on YPD agar, and then patching single colonies onto CSM-Met Hisagar and CSM-Met Leu agar. Clones that grew on CSM-Met His agar but noton CSM-Met Leu agar were cured (i.e., had lost the plasmid pRS425ADS).One such clone was designated EPY300. EPY300 was transformed withexpression plasmid pRS425-ADS-LEU2d, a plasmid identical to pRS425-ADSexcept that instead of LEU2 it contains a LEU2d selection marker (Erhartand Hollenberg (1983) J. Bacteriol. 156: 625-635) yielding host strainY185.

Y185 host cell transformants were selected on synthetic defined media,containing 2% glucose and all amino acids except histidine, leucine, andmethionine (CSM-glucose; MP Biomedicals, Solon, Ohio). The host strainEPY300 is auxotrophic for leucine biosynthesis (leu2), but expressionplasmid pRS425-ADS-LEU2d in Y185 restores leucine prototrophy (LEU2).Single colonies were patched onto selective medium(CSM-glucose-histidine, leucine, methionine), and grown for 2 days. Thecells were scraped from the plate and transferred to 1 mL of 25% (v/v)glycerol in a cryotube. The suspension was mixed, and then stored at−80° C.

Seed flasks of host strain Y185 were established by adding a stockaliquot of the strain to a 125 mL flask containing 25 mL of CSM-glucoselacking leucine and methionine, and by growing the cultures overnight.The cultures were used to inoculate at an initial OD₆₀₀ of approximately0.05 a 250 mL baffled flask containing 40 mL of synthetic defined medialacking leucine, and containing 0.2% glucose, 1.8% galactose, and 1 mMmethionine. The culture was incubated at 30° C. on a rotary shaker at200 rpm. Because the presence of glucose in the media prevents inductionof the GAL1 promoter by galactose, amorpha-4,11-diene production was notinduced until the cells had used up the glucose in the media and hadswitched to using galactose as their main carbon source. At the time ofinoculation, the cultures were overlain with 8 mL of an organic overlayto capture the amorpha-4,11-diene. Samples were taken at 72 hours bytransferring 5 uL of the organic solvent layer to a clean glass vialcontaining 500 uL ethyl acetate containing a known concentration ofbeta- or trans-caryophyllene as an internal standard.

The organic overlay/ethyl acetate samples were analyzed on aHewlett-Packard 6890 gas chromatograph/mass spectrometer (GC/MS) asdescribed in Example 10.

After 72 hours of growth, 3 yeast cultures were found to produce 60.68,54.48, and 59.25 mg/L amorpha-4,11-diene.

Example 18

This example describes the production of amorpha-4,11-diene in anSaccharomyces cerevisiae host strain where the host strain includes anative mevalonate pathway as well as a heterologous mevalonate pathwaythat is under control of a heterologous regulatory control.

Yeast strains CEN.PK2-1C(Y002) (MATA; ura3-52; trp1-289; leu2-3, 112;his3Δ1; MAL2-8C; SUC2) and CEN.PK2-1D (Y003) (MATalpha; ura3-52; trp1-289; leu2-3,112; his3Δ1; MAL2-8C; SUC2) (J. P. van Dijken et al.,Enzyme Microb Technol 26, 706 (Jun. 1, 2000) were cultivated in eitherstandard rich medium (YPD) or in defined synthetic medium (D. Rose, F.Winston, P. Heiter, Methods in yeast genetics: a laboratory coursemanual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1990) lacking appropriate nutrients allowing for selection ofintegrative transformants, plasmid retention, and meiotic progeny.

DNA-mediated transformations into S. cerevisiae were conducted using thelithium acetate procedure as described by R. H. Schiestl, R. D. Gietz,Curr Genet. 16, 339 (December, 1989). All gene disruptions andreplacements were confirmed by phenotypic analysis, colony polymerasechain reaction (“PCR”) and sequencing of amplified genomic DNA. PlasmidspAM489-pAM498 were constructed using the pCR 2.1 (Invitrogen, CarlsbadCalif.) and are schematically described by FIGS. 7A-7C and Table 6. TheHISMX marker sequences are described in M. S. Longtine et al., Yeast 14,953 (July, 1998). Propagation of plasmid DNA was performed inEscherichia coli strain DH5α.

TABLE 6 Crick Watson Genetic Strain 5′ HR Gene #1 Promoter Promoter Gene#2 Marker 3′ HR pAM489 TRP1 tHMGR GAL1 GAL10 ERG20 TRP1 TRP1 pAM490 TRP1tHMGR CUP1 CUP1 ERG20 TRP1 TRP1 pAM491 URA3 tHMGR GAL1 GAL10 ERG13 URA3URA3 pAM492 URA3 IDI1 CUP1 CUP1 tHMGR URA3 URA3 pAM493 ADE1 tHMGR GAL1GAL10 IDI1 ADE1 URA3 pAM494 ADE1 tHMGR CUP1 CUP1 IDI1 ADE1 ADE1 pAM495HIS3 ERG12 GAL1 GAL10 ERG10 HISMX HIS3 pAM496 HIS3 ERG12 CUP1 CUP1 ERG10HISMX HIS3 pAM497 LEU2 ERG19 GAL1 GAL1 ERG8 HISMX LEU2 pAM498 LEU2 ERG19CUP1 CUP1 ERG8 HISMX LEU2

S. cerevisiae strains Y002 and Y003 were prepared for introduction ofinducible mevalonate pathway genes by the following. The ERG9 promoterwas replaced with the S. cerevisiae MET3 promoter by PCR amplificationof the KanMX-PMET3 region from pAM328 (SEQ ID NO: 43) using primers50-56-pw100-G (SEQ ID NO: 44) and 50-56-pw101-G (SEQ ID NO: 45)containing 45 basepairs of homology to the native ERG9 promoter. 10 μgof the resulting PCR product was transformed into exponentially growingY002 and Y003 strains using 40% w/w polyethelene glycol 3350(Sigma-Aldrich St Louis, Mo.), 100 mM lithium acetate (Sigma), 10 μgSalmon Sperm DNA (Invitrogen) and incubation at 30° C. for 30 minutesfollowed by a 42° C. heat shock for 30 minutes (as described by Schiestl& Gietz, Curr. Genet. 16: 339 (1989)). Positive recombinants wereidentified by their ability to grow on rich medium containing 0.5 μg/mlGeneticin (Invitrogen Co, Carlsbad, Calif.) and confirmed by diagnosticPCR. The resultant clones were given the designation Y93 (MAT A) and Y94(MAT alpha). Next, the ADE1 open reading frame was replaced with theCandida glabrata LEU2 gene (CgLEU2). The 3.5 KB CgLEU2 genomic locus wasamplified from C. glabrata genomic DNA (ATCC, Manassas, Va.) usingprimers 61-67-CPK066-G (SEQ ID NO: 46) and 61-67-CPK067-G (SEQ ID NO:47) containing 50 basepairs of flanking homology to the ADE1 openreading frame (ORF). 10 μg of the resulting PCR product was transformedinto exponentially growing Y93 and Y94 as described above. ade1-strainswere selected for growth in the absence of leucine supplementation andconfirmed by diagnostic PCR. The resultant clones were given thedesignation Y176 (MAT A) and Y177 (MAT alpha).

To generate S. cerevisiae strain Y188, 2 μg's of plasmid DNA from pAM491(SEQ ID NO: 48) and pAM495 (SEQ ID NO:49), respectively, were digestedovernight with PmeI (New England Biolabs, Beverly, Mass.) and introducedinto exponentially growing Y176 as described above. Positiverecombinants were selected for by growth on medium lacking uracil andhistidine. Integration into the correct genomic locus was confirmed bydiagnostic PCR.

To generate S. cerevisiae strain Y189, 2 μg's of plasmid DNA from pAM489(SEQ ID NO: 50) and pAM497 (SEQ ID NO: 51), respectively, were digestedovernight with PmeI and introduced into exponentially growing Y177 asdescribed above. Positive recombinants were selected for by growth onmedium lacking tryptophan and histidine. Integration into the correctgenomic locus was confirmed by diagnostic PCR.

Approximately 1×10⁷ cells from Y188 and Y189 were mixed on a YPD mediumplate for 6 hours at room temperature to allow for mating. The mixedcell culture was then plated to medium lacking histidine, uracil andtryptophan to select for growth of diploid cells. 2 μg of plasmid DNAfrom pAM493 (SEQ ID NO: 52) was digested overnight with PmeI andintroduced into exponentially growing diploid cells as described above.Positive recombinants were selected for by growth on medium lackingadenine. Integration into the correct genomic locus was confirmed bydiagnostic PCR. The resultant strain was given the designation Y238.

To generate haploid strains containing the full complement of introducedgenes,

Y238 was sporulated in 2% potassium acetate and 0.02% raffinose liquidmedium. Approximately 200 genetic tetrads (tetrads are four-sporedmeiotic products) were isolated using a Singer Instruments MSM300 seriesmicromanipulator (Singer Instrument Co, LTD. Somerset, UK). Independentgenetic isolates containing the appropriate complement of introducedgenetic material were identified by their ability to grow in the absenceof adenine, histidine, uracil, and tryptophan. Integration of allintroduced DNA was confirmed by diagnostic PCR. The resultant strainswere given the designation Y210 (MAT A) and Y211 (MAT alpha).

2 μg of plasmid DNA from pAM426 (SEQ ID NO:53), containing S. cerevisiaecondon optimized Amorphadeine Synthase (ADS) expressed from the S.cerevisiae GAL1 promoter, was introduced into exponentially growing Y210and Y211 as described above. S. cerevisiae strains that contained thepAM426 plasmid were selected for by their ability to grow in the absenceof leucine supplementation. The resultant strains were given thedesignation Y225 (MAT A) and Y227 (MAT alpha).

2 μg of plasmid DNA from pAM322 (SEQ ID NO: 54), containing S.cerevisiae condon optimized Amorphadeine Synthase (ADS) and cytochromeP450 monooxygenase (AMO) expressed from the S. cerevisiae GAL1 and thecytochrome P450 oxidoreductase (CPR) expressed from the S. cerevisiaeGAL10 promoter, was introduced into exponentially growing Y210 and Y211as described above. S. cerevisiae strains that contained the pAM322plasmid were selected for by their ability to grow in the absence ofleucine supplementation. The resultant strains were given thedesignation Y222 (MAT A) and Y224 (MAT alpha).

Example 19

This example describes the production of α-farnesene or β-farnesene inEscherichia coli host strains.

Seed cultures of host strains B552 and B592 were established by adding astock aliquot of each strain to a 125 mL flask containing 25 mL M9-MOPS,0.8% glucose, 0.5% yeast extract, and antibiotics as detailed in Table1, and by growing the cultures overnight.

The seed cultures were used to inoculate at an initial OD₆₀₀ ofapproximately 0.05, 250 mL flasks containing 40 mL M9-MOPS, 2% glucose,0.5% yeast extract, and antibiotics. Cultures were incubated at 30° C.on a rotary shaker at 250 rpm until they reached an OD₆₀₀ ofapproximately 0.2, at which point the production of α-farnesene orβ-farnesene in the host cells was induced by adding 40 uL of 1 M IPTG.At the time of induction, the cultures were overlain with 8 mL of anorganic overlay to capture the α-farnesene. Samples were taken every 24hours up to 120 hours (total of 5 time points) by transferring 2 uL to10 uL of the organic overlay layer to a clean glass vial containing 1 mLethyl acetate spiked with trans-caryophyllene as an internal standard.In addition, 1 mL aliquots of the cultures were spun down, cell pelletswere resuspended in 250 uL sterile water, and the cell suspensions weretransferred to a glass vial containing 1 mL ethyl acetate spiked withtrans-caryophyllene as an internal standard. In addition, 0.5 mLaliquots of the whole culture broth were added to a glass vialscontaining 1 mL ethyl acetate spiked with trans-caryophyllene as aninternal standard. The whole culture broth samples were extracted in theethyl acetate by vortexing the glass vials for 10 minutes, after which600 uL of the ethyl acetate extraction was transferred to a clean glassvial.

The organic overlay/ethyl acetate samples and the ethylacetate-extracted whole culture broth samples were analyzed on anAgilent 6890N gas chromatograph equipped with an Agilent 5975 massspectrometer (GC/MS) in full scan mode (50-500 m/z). To expedite runtimes, the temperature program and column matrix was modified to achieveoptimal peak resolution and the shortest overall runtime. A 1 uL samplewas separated using a HP-5MS column (Agilent Technologies, Inc., PaloAlto, Calif.) and helium carrier gas. The temperature program for theanalysis was as follows: 150° C. hold for 3 minutes, increasingtemperature at 25° C./minute to a temperature of 200° C., increasingtemperature at 60° C./minute to a temperature of 300° C., and a hold at300° C. for 1 minute. Previous mass spectra demonstrated that theβ-farnesene synthase product was β-farnesene, and that β-farnesene had aretention time of 4.33 minutes using this GC protocol. Farnesene titerswere calculated by comparing generated peak areas against a quantitativecalibration curve of purified β-farnesene (Sigma-Aldrich ChemicalCompany, St. Louis, Mo.) in trans-caryophyllene-spiked ethyl acetate.

Host strain B592 produced approximately 400 mg/L of α-farnesene at 120hours (averaged over 3 independent clones), and had a maximal specificproductivity of approximately 46 mg/L/OD₆₀₀. Host strain B552 producedapproximately 1.1 g/L of β-farnesene at 120 hours (averaged over 3independent clones), and had a maximal specific productivity ofapproximately 96 mg/L/OD₆₀₀ (1 representative clone).

Example 20

This example describes the production of β-farnesene via the DXP pathwayin an Escherichia coli host strain.

Seed cultures of host strains B650, B651, B652, and B653 wereestablished by adding a stock aliquot of each strain to separate 125 mLflasks containing 25 mL M9-MOPS and antibiotics as detailed in Table 1,and by growing the cultures overnight.

The seed cultures were used to inoculate at an initial OD₆₀₀ ofapproximately 0.05 separate 250 mL flasks containing 40 mL M9-MOPSminimal medium, 45 ug/mL thiamine, micronutrients, 1.00E-5 mol/L FeSO4,0.1 M MOPS, 0.5% yeast extract, 20 g/L of D-glucose, and antibiotics.The cultures were incubated at 30° C. in a humidified incubating shakerat 250 rpm until they reached an OD₆₀₀ of 0.2 to 0.3, at which point theproduction of β-farnesene in the host cells was induced by adding 40 uLof 1 M IPTG to the culture medium. At the time of induction, thecultures were overlain with 8 mL of an organic overlay to capture theβ-farnesene. Samples were taken at various time points by transferring100 uL samples of the upper organic overlay layer to a clean tube. Thetube was centrifuged to separate out any remaining cells or media, and10 uL of the organic overlay samples were transferred into 500 uL ethylacetate spiked with beta- or trans-caryophyllene as an internal standardin clean glass GC vials. The mixtures were vortexed for 30 seconds, andthen analyzed as described in Example 18. Escherichia coli host strainB653 produced approximately 7 mg/g DCW β-farnesene.

Example 21

This example describes the production of α-farnesene or β-farnesene in aSaccharomyces cerevisiae host strain. Strain EPY300 was generated byremoving the expression plasmid from Saccharomyces cerevisiae strainEPY224 (Ro et al. (2006) Nature 440: 940-943; PCT Patent PublicationWO2007/005604) by culturing in rich medium. Strain EPY300 was thentransformed with expression plasmids pRS425-FSA or pR425-FSB, yieldinghost strains Y166 and Y164, respectively.

Host cell transformants were selected on synthetic defined media,containing 2% glucose and all amino acids except leucine (SM-glu). Thehost strain EPY300 was auxotrophic for leucine biosynthesis (leu2), butexpression plasmid pRS425-FSA or pRS425-FSB restores leucine prototrophy(LEU2). Single colonies were transferred to culture vials containing 5mL of liquid SM-glu lacking leucine. The cultures were incubated byshaking at 30° C. until growth reaches stationary phase. The cells werestored at −80° C. in cryo-vials in 1 mL frozen aliquots made up of 400μL 50% glycerol and 600 μL liquid culture.

Seed cultures were established by adding a stock aliquot to a 125 mLflask containing 25 mL SM-glu lacking leucine, and growing the culturesovernight. The seed cultures were used to inoculate at an initial OD₆₀₀of approximately 0.05 250 mL baffled flasks containing 40 mL ofsynthetic defined media lacking leucine, 0.2% glucose, and 1.8%galactose. Cultures were incubated at 30° C. on a rotary shaker at 200rpm. Because the presence of glucose in the media prevents induction ofthe Gall promoter by galactose, farnesene production was not induceduntil the cells use up the glucose in the media and switch to usinggalactose as their main carbon source. The cultures are overlain with 8mL methyl oleate or isopropyl myristate. Samples were taken once every24 hours by transferring 2-10 uL of the organic solvent layer to a cleanglass vial containing 500 uL ethyl acetate containing a knownconcentration of beta- or trans-caryophyllene as an internal standard.In addition, 0.5 mL aliquots of the whole culture broth were added to aglass vials containing 1 mL ethyl acetate spiked withtrans-caryophyllene as an internal standard. The whole culture brothsamples were extracted in the ethyl acetate by vortexing the glass vialsfor 10 minutes, after which 600 uL of the ethyl acetate extraction wastransferred to a clean glass vial.

Host strain Y166 produced approximately 9.8 mg/L of α-farnesene at 120hours (averaged over 3 independent clones), and had a maximal specificproductivity of approximately 3 mg/L/OD₆₀₀ (1 representative clone).Host strain Y164 produced approximately 56 mg/L of β-farnesene at 120hours (averaged over 3 independent clones), and had a maximal specificproductivity of approximately 20 mg/L/OD₆₀₀ (1 representative clone).

Example 22

This example describes the production of γ-terpinene, α-pinene, andterpinolene in Escherichia coli host strains.

Seed cultures of host strains for production of γ-terpinene (E. coliDH1-T1r [pMevT, pMevB-Gpps, pAM445]), α-pinene (E. coli DH1-T1r [pMevT,pMevB-Gpps, pAM443 or pAM442]) or terpinolene (E. coli DH1-T1r [pMevT,pMevB-Gpps, pAM444] were established by adding a stock aliquot of eachstrain to separate 125 mL flasks containing 25 mL M9-MOPS, 2% glucose,0.5% yeast extract, and antibiotics as detailed in Table 1, and bygrowing the cultures overnight to late exponential phase.

The seed cultures were used to inoculate at an initial OD₆₀₀ ofapproximately 0.05, 250 mL flasks containing 40 mL M9-MOPS, 2% glucose,0.5% yeast extract, and antibiotics. At time of inoculation, thecultures were also overlain with 4 mL hexadecane. Cultures wereincubated at 30° C. on a rotary shaker at 200-250 rpm until they reachedan OD₆₀₀ of approximately 0.2, at which point the production of thecompound of interest in the host cells in the host cells was induced byadding 40 uL of 1 M IPTG. Samples were taken once per day for 96 hoursby transferring 200 uL of the hexadecane layer to a 0.6 mL microfugetube. For analysis, the hexadecane overlay was diluted 1:1 or 1:10 withethyl acetate spiked with trans-caryophyllene as an internal standard ina 1.8 mL GC vial. In addition, 1 mL aliquots of the cultures were spundown, cell pellets were resuspended in 250 uL sterile water, and thecell suspensions were transferred to a glass vial containing 1 mL ethylacetate spiked with trans-caryophyllene as an internal standard. Thecell pellets were extracted in the ethyl acetate by vortexing the glassvials for 15 minutes, after which 500 uL of the ethyl acetate extractionwas transferred to a clean glass vial.

The hexadecane/ethyl acetate samples and the ethyl acetate-extractedcell pellet samples were analyzed on an Agilent 6890N gas chromatographequipped with an Agilent 5975 mass spectrometer (GC/MS) in full scanmode (50-500 m/z). To expedite run times, the temperature program andcolumn matrix was modified to achieve optimal peak resolution and theshortest overall runtime. A 1 μL sample was split (a split ratio between1:2 and 1:50 was selected based on sample concentration) and thenseparated using a HP-5MS column (Agilent Technologies, Inc., Palo Alto,Calif.) and helium carrier gas. The temperature program for the analysiswas as follows: 75° C. hold for 3 minutes, increasing temperature at 20°C./minute to a temperature of 115° C., increasing temperature at 60°C./minute to a temperature of 300° C., and a hold at 300° C. for 0.5minute. The various products, γ-terpinene, α-pinene, and terpinolenewere observed at 5.4, 4.1, 5.4, and 5.9 minutes, respectively. Titerswere calculated by comparing generated peak areas against a quantitativecalibration curve of purified standards in trans-caryophyllene-spikedethyl acetate.

Example 23

This example describes the production of linalool, limonene, β-pinene,β-phellandrene, carene, or sabinine in Escherichia coli host strains.

Seed cultures are established by adding a stock aliquot of each strainto separate 125 mL flasks containing 25 mL M9-MOPS, 0.5% yeast extract,2% glucose, and antibiotics as detailed in Table 1, and by growing thecultures overnight.

The seed cultures are used to inoculate at an initial OD₆₀₀ ofapproximately 0.05, 250 mL baffled flasks containing 40 mL M9-MOPS, 0.5%yeast extract, 2% glucose, and antibiotics. Cultures are incubated at30° C. on a rotary shaker at 250 rpm until they reach an OD₆₀₀ ofapproximately 0.2, at which point the production of the compound ofinterest in the host cells is induced by adding 40 ul of 1 M IPTG to theculture medium. The compound of interest is separated from the culturemedium through solvent-solvent extraction, or by settling anddecantation if the titer of the compound of interest is large enough tosaturate the media and to form a second phase.

What is claimed is:
 1. A method for producing an isoprenoid comprising:(a) obtaining a plurality of bacterial or fungal host cells thatcomprise a heterologous nucleic acid encoding one or more enzymes of amevalonate pathway for making isopentenyl pyrophosphate, whereinexpression of the one or more enzymes is under control of at least oneheterologous transcriptional regulator, and wherein said mevalonatepathway comprises (i) an enzyme that condenses acetoacetyl-CoA withacetyl-CoA to form HMG-CoA; (ii) an enzyme that converts HMG-CoA tomevalonate; (iii) an enzyme that phosphorylates mevalonate to mevalonate5-phosphate; (iv) an enzyme that converts mevalonate 5-phosphate tomevalonate 5-pyrophosphate; and (v) an enzyme that converts mevalonate5-pyrophosphate to isopentenyl pyrophosphate; and (b) culturing the hostcells in a medium wherein temperature is maintained at a level whichwould provide for about 90% or less of a maximum specific growth ratefor the plurality of bacterial or fungal host cells.
 2. The method ofclaim 1, wherein the at least one heterologous transcriptional regulatoris inducible.
 3. The method of claim 1, wherein the pathway enzymes areunder control of a single transcriptional regulator.
 4. The method ofclaim 1, wherein the pathway enzymes are under control of a multipletranscriptional regulator.
 5. The method of claim 1, wherein the hostcells are Escherichia coli.
 6. The method of claim 1, wherein the hostcells are yeast cells.
 7. The method of claim 1, wherein the host cellsare Saccharomyces cerevisiae.
 8. The method of claim 1, wherein thepathway comprises a nucleic acid sequence encoding a mevalonate pathwayenzyme from a prokaryote having an endogenous mevalonate pathway.
 9. Themethod of claim 8, wherein the prokaryote is of the genus selected fromEnterococcus, Pseudomonas, and Staphyloccoccus.
 10. The method of claim9, wherein the nucleic acid sequence is selected from acetyl-CoAthiolase, HMG-CoA synthase, HMG-CoA reductase, and mevalonate kinase.11. The method of claim 8, wherein the nucleic acid sequence encodes aClass II HMG reductase.
 12. The method of claim 1, wherein thetemperature of the medium is at least about 2-20° C. below that whichwould provide for the maximum specific growth rate.
 13. The method ofclaim 12, wherein the host cells are cultured at a temperature at leastabout 5° C. below that which would provide for the maximum specificgrowth rate.
 14. The method of claim 12, wherein the temperature of themedium is at least about 10° C. below that which would provide for themaximum specific growth rate.
 15. The method of claim 1, wherein themedium comprises a carbon source and the amount of carbon source willprovide for about 90% or less of the maximum specific growth rate. 16.The method of claim 15, wherein the amount of the carbon source willprovide for about 75% or less of the maximum specific growth rate. 17.The method of claim 15, wherein the amount of the carbon source willprovide for about 50% or less of the maximum specific growth rate. 18.The method of claim 15, wherein the amount of the carbon source willprovide for about 25% or less of the maximum specific growth rate. 19.The method of claim 15, wherein the amount of the carbon source willprovide for about 75%-10% of the maximum specific growth rate.
 20. Themethod of claim 1, wherein the medium comprises a nitrogen sourcepresent in an amount below that which would provide for about 90% orless of the maximum specific rate.
 21. The method of claim 20, whereinthe amount of the nitrogen source will provide for about 75% or less ofthe maximum specific growth rate.
 22. The method of claim 20, whereinthe amount of the nitrogen source will provide for 50% or less of themaximum specific growth rate.
 23. The method of claim 20, wherein theamount of the nitrogen source will provide for about 25% or less of themaximum specific growth rate.
 24. The method of claim 20, wherein theamount of the nitrogen source will provide for about 75%-10% of themaximum specific growth rate.
 25. The method of claim 1, wherein theisoprenoid is produced in an amount greater than about 10 grams perliter of medium.
 26. The method of claim 1, wherein the isoprenoid isproduced in an amount greater than about 50 mg per gram of dry cellweight.
 27. The method of any one of claims 25 or 26, where the amountof isoprenoid is produced in less than about 150 hours.
 28. The methodof any one of claims 25 or 26, where the amount of isoprenoid isproduced in less than about 96 hours.
 29. The method of any one ofclaims 25 or 26, where the amount of isoprenoid is produced in less thanabout 72 hours.
 30. The method of claim 1, wherein the isoprenoid isselected from the group consisting of a hemiterpene, monoterpene,diterpene, triterpene, tetraterpene, and polyterpene.
 31. The method ofclaim 1, wherein the isoprenoid is not a carotenoid.
 32. The method ofclaim 1, wherein the isoprenoid is a C₅-C₂₀ isoprenoid.
 33. The methodof claim 1, wherein the isoprenoid is selected from the group consistingof abietadiene, amorphadiene, carene, α-farnesene, β-farnesene,farnesol, geraniol, geranylgeraniol, isoprene, linalool, limonene,myrcene, nerolidol, ocimene, patchoulol, β-pinene, sabinene,γ-terpinene, terpinolene and valencene.